
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12889-7
10.1016/j.heliyon.2024.e36858
e36858
Research Article
Molecular characterization of superbugs K. pneumoniae harboring extended-spectrum β-lactamase (ESBL) and carbapenemase resistance genes among hospitalized patients in southwestern Iran, Western Asia
Moradi Farhad f.moradi1993@gmail.com
ab
Akbari Maryam c
Vakili-Ghartavol Roghayyeh d
Ostovari Mohsen e
Hadi Nahal nahalhadi@gmail.com
hadina@sums.ac.ir
b⁎
a Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
b Department of Bacteriology & Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
c Maryam Akbari, Department of Microbiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
d Roghayyeh Vakili-Ghartavol: Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
e Mohsen Ostovari, Department of Medical Physics and Biomedical Engineering, Shiraz University of Medical Sciences, Shiraz, Iran
⁎ Corresponding author. Department of Bacteriology & Virology, School of Medicine, Shiraz University of Medical Sciences, Zand St, Imam Hossein Sq, Shiraz, Iran. nahalhadi@gmail.comhadina@sums.ac.ir
24 8 2024
15 9 2024
24 8 2024
10 17 e3685816 5 2024
22 8 2024
23 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Background

Detection of K. pneumoniae superbugs carrying Extended-spectrum β-lactamase (ESBL) and Carbapenemase resistance genes among hospitalized patients is crucial for infection control and prevention. The aim of this molecular study was to investigate the spread of ESBL and Carbapenemase-producing K. pneumoniae in two hospitals located in Southwest Iran.

Methods

One hundred clinical isolates of K. pneumoniae were randomly collected from two hospitals over a period of five months, from November 2023. The isolates were confirmed using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae isolates that exhibited resistance to at least one of the three indicator cephalosporins or carbapenems were selected for evaluation of ESBL and carbapenemase production. This was done using a combination disk confirmatory test and the modified carbapenem inactivation method (mCIM). Finally, the presence of ESBLs and carbapenemase resistance encoding genes was assessed using PCR and specific primers.

Results

Out of the 100 isolates, the percentage of antibiotic resistance was cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Additionally, thirty isolated strains were found to be multidrug-resistant. Out of these, twenty-seven strains demonstrated a potential for ESBLs, twenty strains for Carbapenemase, and seventeen strains for both ESBLs and Carbapenemase production. Moreover, the occurrence of ESBLs and carbapenemase genes was as follows: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). It is important to mention that we did not detect the blaIMP and blaKPC. resistant genes among clinical isolates.

Conclusion

Based on the results, the existence of this type of resistance in hospital centers needs to be reevaluated in terms of empirical antibiotic prescribing. Additionally, it is recommended that infection control measures should be taken for public health. Also, it's suggested that hospital-acquired infections caused by superbug K. pneumoniae resistant strains should be addressed.

Keywords

Klebsiella pneumoniae
ESBLs
Carbapenemase
Antibiotic resistance
Superbugs
==== Body
pmc1 Introduction

Antibiotic resistance has several negative consequences, such as increased health costs, prolonged hospitalization, and higher mortality rates. Given new drugs are constantly being developed, it is crucial for healthcare professionals to promptly and profoundly modify their prescription and use of antibiotics. Without changes in antibiotic usage behavior, antibiotic resistance will continue to be a significant threat [1,2]. These behavioral changes, such as using antibiotics prophylactically, getting vaccines, practicing good hand hygiene, maintaining sexual hygiene, and practicing safe food handling, can help reduce the transmission and spread of infections, and combat antibiotic resistance. It is important to note that bacteria, being intelligent organisms, can to engage in multiple genetic exchanges, significantly contributing to the development of antibiotic resistance within populations [[1], [2], [3]]. The most important antibiotic resistances that we currently face include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), multi-drug-resistant Mycobacterium tuberculosis (MDR-TB), carbapenemase-producing Enterobacterales (CPE), and extended-spectrum β-lactamase-producing Enterobacteriaceae. Among the Enterobacteriaceae family, ESBL and carbapenemase-producing K. pneumoniae are particularly of importance since they can cause serious nosocomial infections and pose a threat to patients [3,4]. Additionally, the global spread of multidrug-resistant K. pneumoniae presents a major obstacle to clinical patient care, especially in countries with limited access to epidemiological data on antimicrobial resistance. Over the past decade, this bacterial species has evolved into a classical and hyper-virulent pathotypes, confirming its status as a major global pathogen [[2], [3], [4]].

The classical pathotypes of K. pneumoniae cause septicemias, soft tissue infections, hospital-acquired urinary tract infections, or pneumonia in immunocompromised patients. However, hyper-virulent pathotypes can also cause community-acquired infections in healthy, young individuals with normal immunity. Although third- and fourth-generation cephalosporins, and even carbapenems, are commonly used in many medical centers to treat Klebsiella infections, the emergence of ESBLs and carbapenemase-producing enzymes has led to treatment failures. Several resistance enzymes, such as Klebsiella pneumoniae carbapenemase (KPC), New Delhi Metallo-beta-lactamase (NDM-1), and class D carbapenemase (OXA-48), have been identified as the main causes of antibiotic resistance. These enzymes were initially found in K. pneumoniae [5]. Moreover, ESBL-producing K. pneumoniae is known to be more virulent due to its capability to produce isochorismatase, lyase, catalase, and oxidoreductase [5,6]. It should be noted that K. pneumoniae possesses a diverse range of genes that are linked to antibiotic resistance. This particular attribute distinguishes it from other gram-negative organisms that are responsible for opportunistic infections. These bacteria exhibit resistance to commonly employed antibiotics, thereby presenting a challenge in their treatment and increasing the vulnerability to severe infections. Hence, it is crucial to isolate K. pneumoniae strains that produce ESBL and carbapenemase within hospital settings as a part of infection control and prevention measures [6,7]. Therefore, it is necessary to periodically monitor the global prevalence of these strains in order to select new treatment strategies and control the infection caused by drug-resistant strains. Furthermore, existence of ESBL and carbapenemase-producing K. pneumoniae is considered as an epidemiological indicator of colonization. Currently, the K. pneumoniae carbapenemase enzymes are the most significant class A serine carbapenemases and are associated with significant morbidity and mortality [7,8].

Besides, in 2024, the World Health Organization has announced that carbapenem-resistant K. pneumoniae is included in the bacterial priority pathogens list [9]. Given the significance of the prevalence and antibiotic resistance of K. pneumoniae, it is essential to conduct regular monitoring in medical centers with a high patient load at various times [10,11]. Due to the significance of the above mentioned points and the prevalence of Klebsiella in our medical centers, we conducted this study to examine the spread of ESBL- and Carbapenemases-producing K. pneumoniae in two teaching hospitals in southwest Iran, which admit a high number of patients in a short duration. Our aim was to monitor this type of antibiotic resistance in this bacterium to facilitate infection control and establish the optimal antibiotic treatment plan for the future.

2 Material and methods

2.1 Specimen collection and identification

In this study, a total of 100 clinical isolates of K. pneumoniae were randomly collected from the laboratories of two teaching hospitals over a period of five months, starting from November 2023. The geographical map of the study region is shown in Fig. 1. For our study, we collected clinical specimens from various hospital departments, such as the ICU, infection, pediatric, internal, and outpatient units. We isolated and purified K. pneumoniae from different clinical specimens, including urine, shunt, bronchial fluids, blood, and wounds. We randomly selected one hundred hospitalized patients and outpatients to provide specimens (Fig. 1). Furthermore, K. pneumoniae has been identified in a wide range of patient afflictions, encompassing prevalent urinary tract infections (UTIs); cystitis; biliary tract infections; respiratory disorders such as cystic fibrosis, pneumonia, and chronic obstructive pulmonary disease (COPD); bacteremia and sepsis; meningitis; hydrocephalus involving the utilization of Ventriculoperitoneal shunts; and diverse forms of infected wounds such as pressure ulcers, surgical incisions, or abscesses. To phenotypically confirm these clinical isolates, we cultured them on EMB and blood agar media (Merck, Germany). Additionally, we performed standard supplementary tests, including examinations of colony morphology, Gram staining, oxidase, lactose fermentation, TSI, SIM, MR-VP, urease, lysine decarboxylase, arginine dihydrolase, and Simmons citrate cultures (Merck, Germany) for each isolate. Moreover, we genetically confirmed all strains by detecting the capsular polysaccharide synthesis regulating gene (rcsA) using specific primers (Pishgam Co, Iran) and the polymerase chain reaction (PCR) method using ABI Veriti 96 thermal cycler (Table 1).Fig. 1 Geographic map of the study region and rate of K. pneumoniae isolated from infected patients based on gender, different hospital departments, and origin of laboratory specimens.

Fig. 1

Table 1 The primer sequence information and PCR program.

Table 1Gene	Primer Sequence	PCR Product Size (bp)	PCR Program	
Initial Denaturation	Denaturation	Annealing	Initial Extension	Final Extension	PCR Cycle	Ref	
rcsA-F
rcsA-R	5′-GGTCAGCCGAACGATATGAT-3′
3′-ACGGGATATCTGACCAGTCG	537	95 °C
5min	95 °C
1min	57 °C
45sec	72 °C
45sec	72 °C
5min	30	[11]	
blaSHV-F
blaSHV - R	5′-ATGCGTTATATTCGCCTGTG-3′
3′-AGCGTTGCCAGTGCTCGATC-5′	862	95 °C
5min	95 °C
1min	59 °C
45sec	72 °C
45sec	72 °C
5min	30	[13]	
blaTEM-F
blaTEM-R	5′-GAGTATCAACATTTCCGTGTC-3′
3′-TAATCAGTGAGGCACCTTCTC-5′	889	95 °C
5min	95 °C
30sec	56 °C
30sec	72 °C
45sec	72 °C
1min	30	[13]	
blaCTX-M-F
blaCTX-M-R	5′-CGCTTTGCGATGTGCAG-3′
3′-ACCGCGATATCGTTGGT-5′	551	95 °C
5min	95 °C
30sec	59 °C
15sec	72 °C
15sec	72 °C
1min	30	[13]	
blaOXA-48-F
blaOXA-48-R	5′-TTGGTGGCATCGATTATCGG-3′
3′-GAGCACTTCTTTTGTGATGGC-5′	744	95 °C
5min	95 °C
30sec	57 °C
15sec	72 °C
15sec	72 °C
1min	30	[14]	
blaKPC-F
blaKPC-R	5′-ATGTCACTGTATCGCCGTCT-3′
3′-TTTTCAGAGCCTTACTGCCC-5′	893	95 °C
5min	95 °C
1min	56 °C
45sec	72 °C
45sec	72 °C
5min	30	[14]	
blaVIM-F
blaVIM-R	5′-TCCGCCGTTGTCATAATC-3′
3′-AGAAGGAGTCCTCGGTGAG-5′	291	95 °C
5min	95 °C
30sec	55 °C
30sec	72 °C
15sec	72 °C
5min	30	[16]	
blaIMP -F
blaIMP -R	5′-CTACCGCAGCAGAGTCTTTG-3′
3′-AACCAGTTTTGCCTTACCAT-5′	587	95 °C
5min	95 °C
1min	56 °C
45sec	72 °C
45sec	72 °C
5min	30	[17]	

2.2 Antimicrobial susceptibility testing

The disk diffusion method was used to perform antimicrobial susceptibility tests, following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) in 2022 [12]. The resistance of K. pneumoniae clinical isolates to various antibiotics, such as third- and fourth-generation cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, folate pathway inhibitors, and nitrofurans, was also evaluated. To prepare the bacterial suspension, we utilized overnight cultures to create a turbidity adjusted to a 0.5 McFarland standard. The suspension was then spread on Mueller Hinton agar culture medium (Merck, Germany). Additionally, the following antibiotic disks (MASTDISCS®) were added to the medium and incubated at 35 °C for 18 h: cefoxitin (30ug), cefixime (5ug), cefotaxime (30ug), ceftriaxone (30ug), ceftazidime (30ug), cefepime (30ug), imipenem (10ug), meropenem (10ug), gentamicin (10ug), amikacin (30ug), ciprofloxacin (5ug), levofloxacin (5ug), trimethoprim-sulfamethoxazole (1:19), and nitrofurantoin (300ug). The results were then interpreted according to CLSI categories. For quality control, E. coli ATCC25922 and K. pneumoniae ATCC700603 were used as standard strains. K. pneumoniae isolates that were resistant to at least one of the three indicator cephalosporins were selected for the confirmatory testing of ESBL production using the combination disk method. Furthermore, any isolate that was resistant to either imipenem or meropenem, or both, was selected for the carbapenem inactivation method (mCIM).

2.3 Identification of ESBL-producing isolates

Continuing from the previous step, the isolates that showed resistance to at least one of the ceftazidime, cefotaxime, and ceftriaxone disks were examined for ESBL production. The examination was performed using the combination disk confirmatory test method, following the CLSI procedure [12]. In this method, MASTDISCS® ceftazidime (30 μg) and ceftazidime (30 μg)/clavulanic acid (10 μg) disks were placed on Mueller Hinton medium, which had been inoculated with the suspected bacterial suspension. The medium was then incubated at 35 °C for 16–18 h. Afterwards, the diameter of the non-growth zone was measured. If the diameter of the non-growth zone of the ceftazidime/clavulanic acid disk increased by 5 mm or more compared to the ceftazidime disk alone, the strain was identified as an ESBL producer. During these trials, E. coli ATCC 25922 was used as the negative control, while K. pneumoniae ATCC 700603 was used as the positive control.

2.4 Determination of carbapenemase-producing isolates

The carbapenem inactivation method (mCIM) was used to determine the isolates producing carbapenemase enzymes, following the CLSI protocol [12]. In this method, a loopful of colonies from each suspected isolate was taken from overnight blood agar medium (Merck, Germany) and dissolved in 2 ml of Tryptic Soy broth medium (Merck, Germany). The mixture was then vortexed for 10–15 s. Next, a meropenem disk (10 μg) was dropped into the test tube and incubated for 4 h under aerobic conditions at 35 ± 2 °C. Additionally, 15 min before the end of the incubation period, a suspension of E. coli ATCC 25922 adjusted to a 0.5 McFarland standard was inoculated on Mueller Hinton agar and allowed to dry for 3–10 min. After the completion of the incubation time for the test tube, the meropenem disk (MASTDISCS®) was extracted and placed on Mueller Hinton medium containing E. coli ATCC 25922. This plate was then incubated at 35 ± 2 °C for 24-18 h. The inhibition zone around the meropenem disk was subsequently measured in millimeters. Finally, the results were interpreted according to CLSI guidelines. A zone diameter of 6–15 mm or the presence of pinpoint colonies within the 16–18 mm zone was considered carbapenemase-positive. A zone diameter of 16–18 mm or a zone diameter of ≥19 mm with the presence of pinpoint colonies within the zone were considered intermediate. A zone diameter of ≥19 mm indicated a carbapenemase-negative result.

2.5 ESBLs and carbapenemase-related gene assay

To assess the presence of resistant genes in the clinical isolates of K. pneumoniae that were confirmed to produce ESBLs and Carbapenemase, we extracted bacterial genomic DNA using the AllPrep DNA Mini Kit (Qiagen, Inc.) following the manufacturer's instructions. We then used the polymerase chain reaction (PCR) method with the ABI Veriti 96 thermal cycler machine to detect the broad-spectrum β-lactamase genes (blaSHV, blaTEM, blaCTX-M) and carbapenemase enzyme genes (blaOXA-48, blaVIM, blaIMP, blaKPC). The primer sequences (Pishgam Co, Iran) and PCR program for each reaction, including denaturation, annealing temperature, extension, and number of PCR cycles are displayed in Table 1 [11,13,14,16,17]. In our study, each PCR reaction mixture consisted of 25 μl, with 1 μl DNA template, 0.5 μl of each forward and reverse primers (10 pM), 12.5 μl of DNA Polymerase Master Mix RED (Ampliqon Co., Denmark), and 10.5 μl of DNase- and RNase-free water. Finally, the amplified products were identified through 1 % agarose gel electrophoresis with a 50 bp DNA ladder (Pishgam Co, Iran), and the results were analyzed using a Gel Documentation System (ATP, Iran). In the present study, K. pneumoniae ATCC 700603 strain harboring blaTEM, blaSHV, and blaCTX−M genes; P. aeruginosa PO510 strains harboring genes blaVIM and blaIMP genes; and K. pneumoniae KP1514 harboring blaOXA-48 (Pasteur Institute, Iran) were used as positive controls.

3 Statistical analysis

The data were analyzed using SPSS version 20 (Statistical Package for Social Sciences, SPSS Inc, Chicago, IL, USA). Qualitative data are presented as numbers and percentages. The Chi-square test was used to compare the data. Differences with a p-value ≤0.05 were considered statistically significant.

4 Results

4.1 Characteristics of clinical isolates

The characteristics of the clinical isolates are presented in Fig. 1. Among the cases of K. pneumoniae, 34 and 66 were isolated from male and females, respectively. Moreover, a total of 100 cases of K. pneumoniae were collected from various hospital wards, including the ICU, infection, pediatric, internal, and outpatient units. These isolates were obtained from different sources such as outpatients, urine, shunt, bronchial fluid, blood, and wounds (Fig. 1). The highest percentage of K. pneumoniae isolates was found in urine samples (63 %), while the lowest was in shunt specimens (5 %). However, there was no significant difference in the frequency of K. pneumoniae among different types of specimens (p > 0.05) (Fig. 1).

4.2 Antibiotic susceptibility pattern of clinical isolates of K. pneumoniae

Table 2 presents the antibiotic resistance pattern of clinical isolates of K. pneumoniae to various antibiotics, including third and fourth-generation cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, folate pathway inhibitors, and nitrofurans. Out of the 100 isolates, the percentage of antibiotic reFutisistance was as follows: cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), meropenem (17 %), gentamicin (23 %), amikacin (25 %), ciprofloxacin (35 %), levofloxacin (30 %), trimethoprim-sulfamethoxazole (35 %), and nitrofurantoin (25 %). Overall, our study identified 30 strains of multidrug-resistant K. pneumoniae that were resistant to at least three categories of antibiotics. Importantly, there was no statistically significant difference in the antibiotic resistance patterns among the clinical isolates (p > 0.05).Table 2 Antibiotic susceptibility pattern of the clinical isolate of K. pneumoniae.

Table 2
Category	Antibiotic Agents	
cefoxitin (30ug)	cefixime (5ug)	cefotaxime (30ug)	ceftriaxone (30ug)	ceftazidime (30ug)	cefepime (30ug)	imipenem (10ug)	meropenem (10ug)	amikacin (30ug)	gentamicin (10 μg)	ciprofloxacin (5ug)	levofloxacin (5 μg)	nitrofurantoin (300 μg);
Urine Sample	trimethoprim/Sulfamethoxazole 1:19	
Susceptible	71	72	71	75	71	77	80	83	70	75	50	64	22	59	
Intermediate	0	0	5	4	3	1	0	0	5	2	15	6	16	6	
Resistant	29	28	24	21	26	22	20	17	25	23	35	30	25	35	
p value	391/0	433/0	265/0	472/0	424/0	507/0	932/0	115/0	959/0	850/0	757/0	630/0	925/0	748/0	

4.3 Results of ESBLs and carbapenemase confirmatory and resistance genes assays

As mentioned earlier, we assessed the resistance of K. pneumoniae clinical isolates to third and fourth-generation cephalosporins and carbapenems, as well as the presence of ESBLs and mCIM phenotypes. Furthermore, we examined the presence of related genes. Based on our findings, 27 strains exhibited a potential for ESBLs, 20 strains for Carbapenemase, and 17 strains for ESBLs via Carbapenemase production (Fig. 2A). Out of these strains, three were absolute Carbapenemase producers, and ten were absolute ESBL producers. As shown in Fig. 2 B, the frequencies of ESBLs and carbapenemase genes among the 100 K. pneumoniae isolates were as follows: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). It is worth noting that blaIMP and blaKPC carbapenemase genes were not detected in this study. Among these isolates, ten cases were found to harbor only ESBL genes (blaSHV, blaCTX-M, blaTEM), while three cases only had carbapenemase genes (blaOXA-48, blaVIM). Furthermore, 17 clinical specimens were confirmed to contain both ESBLs and carbapenemase genotypes simultaneously. PCR results revealed that out of 30 MDR isolates, 27 carried at least one β-lactamase gene, while 20 cases carried at least one carbapenemase gene. Our results indicated that 27 K. pneumoniae strains which produced ESBL showed resistance to at least one cephalosporin and harbored the blaSHV gene. These strains were primarily isolated from urine (9 cases) and bronchial fluid (7 cases) specimens. Additionally, 20 strains confirmed as carbapenem-resistant K. pneumoniae showed resistance to imipenem and meropenem in disk diffusion tests, as confirmed by mCIM tests. These strains harbored at least one carbapenem-resistant gene, either blaOXA-48 or blaVIM, and were primarily isolated from urine (7 cases) and bronchial fluid (7 cases) specimens (Fig. 2C and Table 3). Furthermore, ESBL-producing multidrug-resistant (MDR) isolates were recovered from 16 female and 11 male patients. In addition, carbapenemase-producing MDR isolates were recovered from 14 female and 7 male patients aged over 50 years. Moreover, the majority of these MDR isolates were found in the intensive care unit (ICU) with 9 cases, and the infections department with 6 cases. These phenotypic and molecular results underscore the significance of superbugs, specifically K. pneumoniae, that carry resistance genes for ESBL and carbapenemases. These superbugs were isolated from respiratory infections such as cystic fibrosis, pneumonia, chronic obstructive pulmonary disease (COPD), and urinary tract infections (UTIs). Importantly, four clinical isolates were cultured from a contaminated shunt, with three of them simultaneously harboring ESBL and carbapenemase resistance genes (Table 3). Indeed, six strains were isolated from infected wounds. Among these, four strains produced ESBLs and harbored blaSHV and blaTEM, while two strains harbored both ESBLs (blaSHV, blaCTX-M, blaTEM) and carbapenemase (blaOXA-48, blaVIM) resistance genes. Additionally, two cases of blood infection caused by K. pneumoniae were identified. One of these cases was completely resistant to carbapenems (harboring blaOXA-48, blaVIM), while the other case was identified as an ESBL producer (harboring blaSHV, blaCTX-M, blaTEM). According to our results, the coexistence rate of ESBLs and carbapenemase-resistant genes among superbug strains was 25 % and 10 %, respectively. The simultaneous coexistence of ESBLs and carbapenemase among superbug strains was 17 % (Fig. 2C and Table 3). Moreover, the PCR results images for ESBLs and carbapenemases resistance encoding genes prepared in Fig. 3 (The full images prepared as supplementary material, Fig. 3A–E). This information highlights the importance of extra intestinal infection caused by K. pneumoniae, which should be considered for infection control and treatment. More detailed information about the detection rate of carbapenem-resistant and ESBL-producing K. pneumoniae is provided and compared in Fig. 2, Fig. 3, and Table 3.Fig. 2 A; Number of ESBLs and carbapenemase resistance strains. B; Frequency of ESBLs and carbapenemase resistance genes among resistant strains. C; Rate of Frequency and Coexistence of ESBLs, Carbapenemase, and ESBLs-Carbapenemase resistance genes detected from K. pneumoniae clinical isolated based on the clinical specimens.

Fig. 2

Table 3 Characteristics of ESBL and carbapenemase-producing K. pneumoniae in Shiraz, South of Iran.

Table 3Rate of resistant strains of K. pneumoniae	Patient
Specimen
No	Source of infection	Gender/age/Department	Phenotypic Confirmatory Test (ESBL/mCIM)
/Antibiotic resistant Panel	ESBL Resistance gene	Carbapenemase Resistance gene	
Absolut ESBLs producer strains;
10 strain	2	Shunt	Woman/42/ICU	+/CAZ, CTX, CRO, CFM, FEP, FOX, GM, AN, CP, SXT.	blaSHV,blaCTX-M,blaTEM	-	
28	Blood	Man/78/Infection	+/CAZ, CTX, CRO, CFM, FEP, FOX, GM, AN, CP.	blaSHV,blaCTX-M,blaTEM	-	
52	Urine	Woman/57/Outpatient	+/CAZ, CTX, CRO, CFM, FEP, GM, NF.	blaSHV,blaCTX-M,blaTEM	-	
3	Bronchial fluid	Man/78/Infection	+/CAZ, CTX, CRO, CFM, FEP, FOX, GM, AN, CP, LVX.	blaCTX-M,blaTEM	-	
56	Wound	Man/72/Infections	+/CAZ, CTX, CRO, CFM, FEP, GM, SXT, NF.	blaSHV,blaCTX-M	-	
29	Urine	Woman/68/ICU	+/CTX, CRO, CFM, GM, LVX, NF.	blaSHV,blaCTX-M	-	
81	Wound	Man/86/Outpatient	+/FEP, GM, SXT.	blaSHV,blaTEM	-	
47	Wound	Woman/69/Infections	+/CAZ, CTX, AN, SXT, NF.	blaSHV,blaTEM	-	
17	Wound	Man/24/ICU	+/CAZ, AN, LVX, SXT.	blaSHV	-	
10	Urine	Man/69/Infection	+/CAZ, CTX, AN, CP, LVX, SXT, NF.	blaSHV	-	
ESBLs via Carbapenemase producer strains;
17 strain	7	Bronchial fluid	Man/60/ICU	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, AN, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48,blaVIM	
21	Wound	Woman/75/ICU	+/CTX, CRO, MEN, AN, CP, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48,blaVIM	
5	Bronchial fluid	Woman/91/ICU	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, AN, CP,	blaSHV,blaCTX-M,blaTEM	blaOXA-48,blaVIM	
27	Urine	Woman/67/Infection	+/CRO, MEN, IMP, AN, LVX, SXT.	blaSHV,blaCTX-M,blaTEM	blaOXA-48,blaVIM	
39	Urine	Woman/22/Internal	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, CP, LVX, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48,blaVIM	
42	Shunt	Man/73/Infections	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, CP, LVX.	blaSHV,blaTEM	blaOXA-48,blaVIM	
62	Urine	Woman/25/Internal	+/CFM, MEN, GM, SXT, NF.	blaCTX-M,blaTEM	blaOXA-48,blaVIM	
6	Bronchial fluid	Woman/55/ICU	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, AN, CP, LVX.	blaSHV,blaCTX-M,blaTEM	blaVIM	
32	Wound	Woman/58/Infection	+/CTX, MEN, IMP, FEP, FOX, GM, AN.	blaSHV,blaTEM	blaVIM	
76	Urine	Woman/48/Outpatient	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, GM, CP, LVX, NF.	blaSHV,blaTEM	blaVIM	
9	Urine	Woman/52/Outpatient	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, AN.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
14	Shunt	Man/69/Infection	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, GM, AN, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
4	Urine	Woman/4/Paediatric	+/CAZ, CTX, CRO, CFM, MEN, IMP, FEP, FOX, AN, CP, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
22	Bronchial fluid	Man/70/ICU	+/CTX, CRO, IMP, AN, CP, SXT, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
23	Bronchial fluid	Woman/55/ICU	+/CAZ, CTX, CRO, GM, IMP, AN, CP, LVX.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
24	Bronchial fluid	Woman/64/Infection	+/CAZ, CTX, CRO, CFM, IMP, FEP, FOX, AN, CP, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
67	Shunt	Man/76/Infections	+/CAZ, IMP, AN, CP, LVX, NF.	blaSHV,blaCTX-M,blaTEM	blaOXA-48	
Absolut Carbapenemase producer strains;
3 strain	50	Bronchial fluid	Man/76/Infections	+/MEN, IMP, GM, CP, LVX, NF.	-	blaOXA-48,blaVIM	
53	Blood	Woman/58/Infections	+/MEN, IMP, GM, CP, LVX, NF.	-	blaOXA-48,blaVIM	
26	Urine	Woman/53/Outpatient	+/MEN, IMP, GM, NF, STX.	-	blaOXA-48,blaVIM	
Abbreviations; Ceftazidime (CAZ), Cefotaxime (CTX), Ceftriaxone (CRO), Cefixime (CFM), Meropenem (MEN), Imipenem (IMP), Cefepime (FEP), Cefoxitin (FOX), Gentamicin (GM), Amikacin (AN), Ciprofloxacin (CP), Levofloxacin (LVX), Trimethoprim-sulfamethoxazole (SXT), Nitrofurantoin (NF).

Fig. 3 ESBLs and carbapenemases resistance encoding genes assayed through PCR and specific primers. Products were identified by 1 % agarose gel electrophoresis with a 50 bp DNA ladder, and the results were analyzed using a gel documentation device. K. pneumoniae ATCC 700603 strain harboring blaTEM, blaSHV,blaCTX−M genes, P. aeruginosa PO510 strains harboring bla-VIM, and K. pneumoniae KP1514 harboring blaOXA-48 gene were taken as positive control. (The full images of PCR gel results prepared as supplementary material, Fig. 3A–E).

Fig. 3

5 Discussion

ESBL-producing and carbapenem-resistant Enterobacteriaceae are included in the priority list of the World Health Organization because there is an urgent need for new antibiotics to treat and control infections caused by these bacteria [9]. Today, there is an emerging presence of ESBL and carbapenemase-producing Gram-negative bacteria in hospitals and medical centers. Among various hospital-acquired illnesses associated with these bacteria, K. pneumoniae is considered a significant health-related infection, accounting for 30 % of cases. It causes a range of infections including urinary tract infections (UTIs), pneumonia, bacteremia, and wound infections [15,18,19]. Most strains of K. pneumoniae are susceptible to third-generation cephalosporins. However, these bacteria have developed significant levels of resistance to antimicrobial drugs. They are inherently resistant to ampicillin due to the presence of plasmid-encoded ampicillin-hydrolyzing β-lactamases, such as the SHV-1 gene. Additionally, with a single point mutation of the blaSHV-1 gene and the production of β-lactamase, they can also become resistant to third- and fourth-generation cephalosporins. Carbapenem-hydrolyzing enzyme-producing strains of K. pneumoniae are mainly found in hospital settings and can hydrolyze monobactams, carbapenems, and cephalosporins. Consequently, several studies have identified K. pneumoniae as one of the most significant multidrug-resistant organisms in the ESKAPE group. The ESKAPE group comprises Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae [[20], [21], [22]]. Although numerous studies have been conducted worldwide, including in our country, Iran, investigation of the prevalence of ESBL and carbapenemase-producing K. pneumoniae in different years and locations, yields significant gaps in the results. The continuous and changing antibiotic resistance patterns of this bacterium, along with the need for new treatment guidelines in various treatment centers, are the main factors contributing to these gaps. Therefore, it is crucial to periodically review the epidemiology and spread of these antibiotic resistance cases. Additionally, addressing this gap is essential because isolating ESBL and carbapenemase-producing K. pneumoniae in hospital settings is critical for infection control and prevention, given the potential dangers associated with these multidrug-resistant organisms [23,24]. We have mentioned that these bacteria possess the capability to produce enzymes that render the commonly used antibiotics ineffective. As a result, the available treatment options become limited and there is an increased risk of healthcare-associated infections. The presence of ESBL and carbapenemase-producing K. pneumoniae strains in hospitals can lead to outbreaks of difficult-to-treat infections, longer hospital stays, higher healthcare costs, and increased mortality rates among affected patients. These bacterial strains can easily spread within healthcare facilities through patient-to-patient transmission, contaminated medical equipment, healthcare workers, and environmental surfaces. It is essential to implement strict infection control measures and surveillance strategies in order to effectively combat the threat posed by ESBL and carbapenemase-producing K. pneumoniae in hospitals. This involves early detection of resistant strains, use of precautions when contacting infected or colonized patients, and adherence to proper hand hygiene practices, and established protocols for environmental cleaning. Also, holding antimicrobial stewardship programs, and conducting targeted screening of high-risk patient populations are important [[23], [24], [25]]. To address the issue of K. pneumoniae isolation in health centers in our province and city, the lack of information regarding the prevalence of ESBLs and carbapenemase-resistant strains of K. pneumoniae, increase in the resistance rate of K. pneumoniae strains to cephalosporins and carbapenem drugs, prescribing cephalosporins and carbapenems commonly in our healthcare setting, and the need for improved treatment procedures, we conducted a snapshot study. This study aimed to investigate the spread of ESBL and carbapenemase-producing K. pneumoniae in two teaching hospitals located in the southwestern region, Western Asia. In this study, we collected 100 clinical isolates of K. pneumoniae from two hospitals and confirmed them using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae clinical isolates with phenotypic resistant to at least one of the three indicator cephalosporins or carbapenem were selected for the evaluation of ESBL and carbapenemase production. We used a combination disk confirmatory test and a modified carbapenem inactivation method (mCIM) for this purpose. Additionally, we assessed the presence of ESBLs and carbapenemase resistance-encoding genes using PCR and specific primers. Among the 100 isolates, the percentage of antibiotic resistance was as follows: cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Our results showed that the highest resistance among the clinical isolates of K. pneumoniae was against cefoxitin (29 %), while the lowest resistance was against meropenem. We also observed and reported the highest occurrence of K. pneumoniae strains producing ESBL and carbapenemase resistance in urine and bronchial fluid specimens. Furthermore, we found that 27, 20, 17, and 30 strains exhibited a potential for ESBLs, carbapenemase, ESBLs-carbapenemase coexistence, and multidrug resistance, respectively. According to our results, the rate of coexistence of ESBLs and carbapenemase resistance genes among superbug strains was 25 % and 10 %, respectively. We also observed a simultaneous coexistence of ESBLs and carbapenemase among superbug strains at a rate of 17 % (Table 3).

Several studies have been conducted in Iran to determine the prevalence of ESBL-Carbapenemase-producing K. pneumoniae in different regions. For instance, two studies conducted in Isfahan and Tehran reported prevalence rates of 60.4 % and 44.2 % for ESBL-producing K. pneumoniae, respectively [26,27]. Other researchers, such as Habibi et al. (2017), Shams et al. (2014), and Ghasemi et al. (2009–2010), reported prevalence rates of ESBL-producing K. pneumoniae strains in three provinces of Iran at 33.3 %, 80.5 %, and 60 %, respectively [[28], [29], [30]]. Furthermore, Jafari et al. (2019) conducted a molecular epidemiology study that documented a prevalence rate of 35 % for carbapenem-resistant K. pneumoniae isolates in Iran [31]. Other studies from around the world have reported varying frequencies of this type of resistance. For instance, in India, the occurrence of ESBL and Amp-C beta-lactamases was reported as 60 % [32], while in Bangladesh, ESBL-producing K. pneumoniae was reported at 6.6 % [33]. Molecular epidemiology research conducted in Zhejiang, China, documented a prevalence increase of carbapenem-resistant K. pneumoniae from 2.5 % in 2008 to 15.8 % in 2018 [34]. Furthermore, the rate of ESBL production was reported 29.6 % in Nepal [35]. In one hospital in Northwest Ethiopia, 19.2 % of Gram-negative bacilli isolates were ESBL producers, and 5.8 % were carbapenemase producers [36]. Overall, the global prevalence of ESBL production has been reported to range from less than 1 %–74 % [37]. In our study, we observed the following frequencies of ESBLs and carbapenemase genes: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). We did not detect the blaIMP and blaKPC resistance genes among clinical isolates. In comparison to other studies conducted in Iran, Taraghian et al. found blaSHV in 90.9 % of cases, blaTEM in 63.6 % of cases, and blaCTX-M in 63.6 % of cases [13]. Sanikhani et al. reported the presence of blaOXA-48 in 53.9 % of cases [38], while Sohrabi et al. found blaSHV in 100 % of cases but did not detect blaOXA-48 and blaKPC genes [39]. These studies highlight the high prevalence of ESBLs in clinical isolates of K. pneumoniae in Iran, which is a trend of concern given the limited treatment options available for ESBL-producing bacteria. Additionally, Mukherjee et al. reported frequencies of 75 % for blaSHV, 71.4 % for blaCTX-M, and 57.1 % for blaTEM in hvKp isolates [40]. Also, Remya et al. [7] reported that these genes were found in 100 %, 77.2 %, and 66.6 % of hvKp isolates, respectively. In Italy, the frequencies of the blaKPC, blaOXA-48, and blaVIM genes were reported as 10.5 %, 10.5 %, and 5.2 %, respectively [41]. Also, in China, the blaKPC gene was found in 7.1 % of the isolates, while Lev et al. [8] reported blaOXA-48 in 38 % of the isolates in Russia [42].

In the other hand, various studies have reported different prevalence rates of carbapenem-resistant K. pneumoniae in different provinces of Iran and other countries compared to our results. For example, a study conducted in Tabriz, northwestern Iran, in 2020 found that blaKPC (72.0 %) was the most prevalent carbapenemase gene among resistant isolates, followed by blaVIM (12.0 %), blaIMP (4.0 %), and blaOXA-48 (4.0 %) genes, with no coexistence of carbapenemase genes detected in any isolate. Similar to our study, blaIMP was not detected in any isolate in Tabriz [43]. Furthermore, in contrast to our result, the blaIMP gene was found in 58.97 % of the isolates in Egypt (2020) and in 7.2 % of isolates in Pakistan (2021), which is not in the same line with our results [44,45]. Also, similar to our report, two studies from Isfahan province, the neighboring province, found that no carbapenem-resistant K. pneumoniae isolates carried blaKPC [46,47]. Another study conducted in Bushehr (2020), located in southern Iran, reported a lower rate of carbapenemase-producing K. pneumoniae (7.9 %), with carbapenemase genes coexisting in 25.0 % of the isolates. This is in contrast to our results, where 20 % of the cases were carbapenemase-producing, and 10 % of them had coexisting genes [48]. Additionally, unlike countries such as China and Brazil, where blaOXA-48 was not detected, other studies conducted in Bushehr (2020), Tehran (the capital of Iran in 2022), and Uganda (2021) reported a prevalence rate of blaOXA-48 of 33.3 %, 67.6 %, and 36.4 %, respectively, which are much higher than the rate observed in our current study (17 %) [[48], [49], [50], [51], [52]]. Furthermore, in comparison to our results (13 %), various prevalence rates have been reported for the blaVIM gene. For example, 12 % in Tabriz, northwestern Iran [43], 3.2 % in K. pneumoniae isolates from Pakistan [45], no detection in Turkey [53], and 84.62 % in K. pneumoniae clinical isolates from Egypt [44]. Due to the critical nature of K. pneumoniae producing carbapenemase in medical centers, the distribution of various carbapenemase genes in different geographical areas can be attributed to several factors. These factors include variations in empirical treatment protocols within healthcare settings, differences in laboratory techniques used to identify resistance, discrepancies in the study population, and the migration of individuals to different countries for trade or treatment. These factors emphasize the importance of understanding and addressing carbapenem-resistant K. pneumoniae. Furthermore, the prevalence rates of ESBL and carbapenemase-producing K. pneumoniae vary between countries due to differences in antibiotic selection practices and improper prophylaxis. One significant finding from these studies is that the use of CLSI confirmatory tests is highly efficient in detecting ESBL and carbapenemase-producing K. pneumoniae, especially in settings where molecular characterization facilities are not available. Currently, there is a great increase in multi-drug-resistant urinary infections, particularly ESBL. To prevent treatment failure, we need to perform antibiotic susceptibility tests on clinical specimens that contain K. pneumoniae infections [43,44]. Both ESBL and carbapenemase-producing K. pneumoniae are significant public health concerns in Iran and Asia. It is crucial to prevent their spread and improve antibiotic stewardship to combat antibiotic resistance. Understanding the frequency rate of ESBL and carbapenemase-producing K. pneumoniae in hospital units can help hospitals develop and implement appropriate infection control measures. This will minimize the spread of infection among patients and assist doctors in selecting the most appropriate antibiotics for treating patients with these infections. It will also allow healthcare providers to monitor the effectiveness of their infection control measures and identify any potential outbreaks or increases in resistance [45]. Knowing the prevalence rate of these types of resistant bacteria in medical laboratory diagnosis has several applications. It can aid in the development of more accurate and sensitive diagnostic tests to identify these pathogens in clinical samples. It can also help establish quality assurance measures to ensure the accuracy and reliability of diagnostic tests. Additionally, it enables specialized antimicrobial susceptibility testing on clinical isolates of these bacteria.

6 Conclusion

Both extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing K. pneumoniae are antibiotic-resistant bacteria that pose a significant threat to public health. Consequently, particular concern is given to carbapenemase-producing bacteria as they are resistant to most antibiotics, including carbapenems, which are considered a last-resort treatment for bacterial infections. Studies conducted in Iran and Asia have demonstrated the prevalence, mechanisms, and epidemiology of ESBL and carbapenemase-producing K. pneumoniae, emphasizing the urgent need for coordinated efforts to address antibiotic resistance in these regions. Our study highlights the importance of monitoring and understanding the resistance rate in order to guide appropriate treatment strategies and antimicrobial stewardship programs in our region. Furthermore, infection control measures are necessary to prevent the spread of resistant strains.

Ethical Approval and Funding

This work is part of a Ph.D. thesis conducted in the department of bacteriology & virology, school of medicine, 10.13039/501100004320 Shiraz University of medical Sciences (SUMS) , Shiraz, Iran, with ethical approval code IR.SUMS.REC.1402.465 and Track code: 284300.

Consent for publication

In this study, consent was obtained from the patients and experimentation on biological samples was conducted according to established ethical guidelines (IR.SUMS.REC.1402.465).

Data availability statement

Data included in article/supp. Material/referenced in article. No additional information is available for this paper.

CRediT authorship contribution statement

Farhad Moradi: Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Software, Writing – original draft, Writing – review & editing. Maryam Akbari: Data curation, Conceptualization, Formal analysis, Investigation, Methodology, Writing – review & editing. Roghayyeh Vakili-Ghartavol: Formal analysis, Data curation, Conceptualization, Methodology, Software. Mohsen Ostovari: Formal analysis, Data curation, Conceptualization, Methodology, Software. Nahal Hadi: Writing – original draft, Investigation, Conceptualization, Project administration, Supervision, Validation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgement

We are extremely grateful for the sample preparation conducted by the laboratories in our teaching hospitals, as well as for the scientific assistance provided by the Department of Bacteriology & Virology at Shiraz University of Medical Sciences.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36858.
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