Background

Klebsiella pneumoniae (K. pneumoniae) is one of the most important causes of urinary, respiratory, sepsis, and wound infections in communities and hospitals [1, 2]. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae are a leading cause of healthcare-associated infections, which are linked to rising medical expenditures and increased morbidity and mortality [3, 4]. The emergence and dissemination of MDR and XDR K. pneumoniae isolates pose a significant threat to infection control programs and, therefore, require immediate attention [4, 5]. Carbapenems are the last-line for treatment to treat serious infections with MDR K. pneumonia [5]. Unfortunately, Carbapenem-resistant K. pneumoniae (CRKP) is on the rise worldwide and is of especial clinical concern globally as such infections are a challenge to treat [6]. In particular, CRKP is considered a difficult-to-treat organism as there are few therapeutic options [3]. Therefore, it also results in high mortality [6]. The most clinically significant carbapenemase genes in CRKP are Ambler class A β-lactamases (blaKPC), class B metallo-β-lactamases (MBLs) (blaNDM and blaVIM), and class D β-lactamases (blaOXA−48) [6]. The release of blaOXA−48 and blaNDM−1 producing K. pneumoniae causes great concern because they have a significant ability to spread [7]. Combination antibiotics often used in the treatment of CRKP include the following: colistin, in combination with tigecycline or rifampin or carbapenem; fosfomycin plus colistin or amikacin; and double-carbapenem antibiotics (a combination of doripenem and ertapenem) [5].

Since the discovery of blaOXA−48 and blaNDM carbapenemases in Turkey and India in 2001 and 2008, respectively, these strains have been implicated in a large number of nosocomial outbreaks in other parts of the world, including the Middle East (e.g., Iran) [3,4,5].

Given the diffusion of different resistances (extended-spectrum β-lactamase (ESBL), MDR, and carbapenem) in K. pneumoniae on both national and international levels and the lack of data on their frequency in this geographical area, we aimed at investigating the frequency of K. pneumoniae and the phenotypic and genotypic level of MDR, XDR, and carbapenem-resistant K. pneumoniae in patients with infections from central Iran.

Materials and methods

Sample collection

A statement to confirm that all methods were carried out in accordance with relevant guidelines and regulations. Must include a sentence confirming that informed consent was obtained from all subjects and/or their legal guardian(s). This study protocol was approved by the Ethics Committee of the Arak University of Medical Sciences (ARAKMU.REC.1396.3.7). For this cross-sectional, descriptive study, 546 clinical samples of urine, tracheal aspirates, wound, and blood were collected from adult inpatients in the Imam Khomeini Hospital (Khomein, Iran) from April 2017 to March 2019. Patients had not received antibiotics before the sampling.

Phenotypic investigation

K. pneumoniae isolates were identified by Gram stain and conventional biochemical tests (Triple Sugar Iron(TSI), Simmons Citrate agar, SIM, Urea agar, Lysine Iron Agar (LIA), Methyl Red / Voges-Proskauer (MR/VP), Oxidative fermentative (OF)), and they were confirmed by application programming interface (API) testing (bioMérieux, France) [8]. K. pneumoniae ATCC 700,603 and Escherichia coli ATCC 25,922 were used as controls in each assay (acquired from the Microbiology Department of the Arak University of Medical Sciences).

Investigating K. pneumoniae antibiotic resistance by disk diffusion

Using the Clinical and Laboratory Standards Institute (CLSI) guidelines [9], an antibiogram assay was performed on the isolated K. pneumoniae colonies. The antibiotic discs contained ampicillin (30 µg), cotrimoxazole (25 µg), cefixime (5 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), gentamicin (10 µg), amikacin (30 µg), tetracycline (30 µg), doxycycline (30 µg), minocycline (30 µg), tigecycline (15 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ampicillin/sulbactam (100/10 µg), piperacillin/tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), doripenem (10 µg), aztreonam (30 µg), colistin (10 µg), and fosfomycin (200 µg) (Mast Diagnostics, United Kingdom) [4, 10].

The minimum inhibitory concentration (MIC) of imipenem, meropenem, and colistin for isolates resistant to carbapenems was determined by using E-test (Liofilchem, Italy) according to the 2021 CLSI guidelines [9].

Detection of ESBL and AmpC β-lactamases by phenotypic methods

To recognize ESBL-positive isolates, the specimens were subjected to combination disk diffusion [11] and double-disk synergy testing procedures [12], and to recognize AmpC-positive isolates, disk testing and phenol boronic acid procedures were used [13].

Detection of carbapenemase

To recognize carbapenemase-positive isolates, the specimens were subjected to the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and EDTA-modified carbapenem inactivation method (eCIM) procedures according to the 2021 CLSI guidelines [9, 14].

Genotypic investigations

DNA extraction

DNA was extracted from the K. pneumoniae isolates using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol.

All culture-positive samples were confirmed to be positive using the 16s RNA primers in the PCR method (Table 1) [15]. The ESBL (blaTEM, blaSHV, and blaCTX−M−1, 2, 8, 15), AmpC (blaCIT, blaCMY−2, blaACC, blaFOX, blaMOX, and blaDHA), and carbapenemase genes [(Ambler class A: blaKPC and blaGES), (Ambler class B: MBLs: blaNDM, blaVIM, blaSIM, blaGIM, blaSPM, and blaIMP) (Ambler class D: blaOxa−48)] were recognized by PCR [16, 17]. Genes sul1 and 2 for sulfonamide resistance and tet(A) and (B) genes for tetracycline resistance were recognized by PCR as well [18]. To amplify the PMQR targets, PCR of the qnr determinant genes qnrA, qnrB, and qnrS was performed [19]. Mutations in the parC and gyrA genes of the quinolone-resistant K. pneumoniae isolates were determined using DNA sequencing [18]. Quaternary ammonium compound (QAC) resistance genes were identified using PCR (Table 1) [18].

Table 1 The primers used in this study
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Integron detection

To check class 1, 2, and 3 integrons, a PCR assay was performed as shown in Table 1.

Results

Phenotypic and genotypic investigation

Out of the 546 infectious samples, 121 (22.1%) were identified as K. pneumoniae using the culture method. All culture-positive samples were confirmed to be positive using the 16s RNA primers in the PCR method (Table 1). The average age of the K. pneumoniae patients was 47 years and 3 months. Of the 121 patients included in the study, 82 (67.7%) males and 39 (32.2%) females were infected by K. pneumoniae, yielding an infection ratio of males to females of 2.1:1. The most infectious samples of K. pneumoniae were seen in urine (62, 51.2%), respiratory (48, 39.6%), wound (11, 9%), and blood infections (8, 6.6%).

Phenotypic and genotypic antibiotic resistance determination

Using the CLSI 2021 guidelines, the highest resistance rates of K. pneumoniae were obtained against ampicillin (119/121, 98.3%), cotrimoxazole (78/121, 64.4%), cefixime (77/121, 63.6%), cefotaxime (77/121, 63.6%), ceftriaxone (77/121, 63.6%), ceftazidime (77/121, 63.6%), and gentamicin (74/121, 61.1%) (Table 2). Of the K. pneumoniae isolates, 77 were ESBL-positive. All ESBL-positive isolates contained blaTEM resistance genes (Table 2). The most frequent ESBL genes were blaTEM (77, 100%), blaCTX−M1 (76, 98.7%), blaSHV (76, 98.7%), and blaCTX−M15 (73, 94.8%). The most frequent AmpC genes were blaCIT (28, 48.3%) and blaCMY−2 (26, 44.8%) (Table 2).

Table 2 Phenotypic and Genotypic antibiotic resistance rates in K. pneumoniae
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Out of 121, 52 (43%) showed resistance to ciprofloxacin and levofloxacin. Among the PMQR determinants, qnrB and qnrS were positive in 25 (48%) and 19 (36.5%) of the isolates, respectively. Of the K. pneumoniae isolates, 63 (52%) exhibited MDR. Of the isolates harboring PMQR, 51.9% had the same mutations in gyrA at amino acid 83 (replacement of serine with leucine) and 0% in parC at amino acid 80 (replacement of serine with isoleucine; GenBank accession no. HM068910). Class 1 integrons (97, 80.1%) were the most frequent integron class (Table 2).

Carbapenem-resistant K. pneumoniae (CRKP) isolates

Overall, 51 (42.1%) of the CRKP isolates were resistant to carbapenems, 36 (70.5%) of which were MBL-positive. The average age of the CRKP patients was 64 and a half years. The most infectious CRKP samples were seen in respiratory (33, 64.7%), wound (8, 15.7%), urine (6, 11.7%), and blood infections (4, 7.8%). The isolates were all resistant to ampicillin, cotrimoxazole, cefixime, cefotaxime, ceftriaxone, ceftazidime, imipenem, and ertapenem (Table 3).

Table 3 Phenotypic and Genotypic antibiotic resistance rates in CRKP isolates
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All these isolates displayed MDR. The most common ESBL resistance genes were blaTEM (51, 100%), blaCTX−M−1 (51, 100%), blaSHV (51, 100%), and blaCTX−M−15 (49, 96%). The most abundant AmpC resistance genes were blaCIT (28, 54.9%) and blaCMY−2 (26, 51%). The most common carbapenemase resistance genes were blaOxa−48 and blaNDM in 46/51 (90.1%) and 36/51 (70.5%) of the CRKP isolates, respectively (Table 3). A total of 31 isolates (60.7%) of CRKP were found with both blaOxa−48 and blaNDM genes together. Of the CRKP isolates, 3.9% were resistant to tigecycline. Amikacin was observed to be the most active factor among the aminoglycoside family of antibiotics. Tigecycline was the most effective antibiotic, with 96.1% susceptibility. BlaNDM-positive isolates were resistant to the most antibiotics, and all of them contained blaTEM, blaCTX−M1, blaSHV, and blaCTX−M15 resistance genes (Table 3).

Discussion

These isolates were highly resistant to ampicillin (93.8%) and cotrimoxazol (64.4%) but least resistant to tigecycline (1.6%), colistin (3.3%), and fosfomycin (4.1%). In other studies in southern and northern Iran, K. pneumoniae isolates were most resistant to cefazolin (45.9%) and ampicillin/sulbactam (93%), respectively [23, 24]. In prior research in Tehran and southern Iran, these isolates showed the lowest antibiotic resistance to tigecycline (9% and 3.5%, respectively) [5, 23].

In this study, 64.4% of the K. pneumoniae isolates were resistant to cotrimoxazole, and the most abundant sulfonamide gene was sul1 (70.5%). In other studies in Hamedan and Shahrekord, resistances to cotrimoxazole of 52% and 63%, respectively, were seen in K. pneumoniae isolates; the most abundant sulfonamide genes were sul1 (60.9%) and sul2 (60.6%), respectively [25, 26]. In this study, all CRKP isolates were resistant to cotrimoxazole, and all of these isolates exclusively contained sul1 genes. In a study in Turkey, 86.3% resistance to cotrimoxazole was observed in CRKP isolates, and the most abundant sulfonamide gene was sul1 (100%) [27].

It seems likeliest that the high use of cotrimoxazole in infections and the spread of resistance genes between bacterial strains have caused high resistance to cotrimoxazole and sulfonamides.

The genes most commonly associated with ESBL in K. pneumoniae isolates are blaCTX−M, blaTEM, and blaSHV [28]. In this research, 63.6% of the K. pneumoniae isolates were ESBL, and the most common ESBL genes were blaTEM (100%), blaCTX−M1 and blaSHV (98.7%), and blaCTX−M15 (94.8%). In a study in Azerbaijan, 68% of the K. pneumoniae isolates were ESBL, and the most common ESBL genes were blaSHV (58%) and blaCTX−M15 (55%). The widespread use of multiple β-lactam agents in recent years has led to the advent of ESBLs, which are frequent carriers of the genes blaTEM and blaSHV [24].

In this research, all CRKP isolates were 100% ESBL positive, and all of these isolates contained the genes blaTEM, blaCTX−M1, blaSHV (100%), and blaCTX−M15 (96%). In other studies in Isfahan and northern Iran, 97.9% and 64% of CRKP isolates were ESBL positive, respectively, and the most abundant ESBL genes in these isolates were blaCTX−M15 (97.9%) and blaSHV (91.4%), respectively [6, 24].

In this research, 96% of the CRKP isolates shared four of the same genes: blaTEM, blaCTX−M1, blaSHV, and blaCTX−M15. In another study, 93.6% of CRKP isolates shared three genes: blaTEM, blaSHV, and blaCTX−M15 [6]. These resistances are usually located on mobile genetic elements, such as plasmids, which are easily transferable within and between bacterial species. In the Middle East, studies have shown that the frequency of ESBL in K. pneumoniae has increased over the last 10 years [28].

In this report, 47.9% of the K. pneumoniae isolates were AmpC positive, and the most abundant genes were blaCIT (48.3%) and blaCMY−2 (44.8%). In other studies, 9% and 19% of isolates were AmpC positive, and the most abundant AmpC genes were blaMOX and blaCMY, respectively [29, 30].

In this report, all CRKP isolates were (100%) AmpC positive, and the most abundant genes were blaCIT (54.9%) and blaCMY−2 (51%). In other studies, 100% and 45.9% of CRKP isolates were AmpC positive, and the most abundant gene was blaDHA (100%) and blaDHA (45.9%), respectively [31, 32].

MDR bacteria such as MDR K. pneumoniae is a common nosocomial pathogen that has become a global public health concern due to difficult-to-treat infections [2, 3, 33, 34]. In this investigation, 52% and 11.6% of the K. pneumoniae isolates were MDR and XDR positive, respectively. In other studies, 58% and 13% of K. pneumoniae isolates were MDR and XDR positive, respectively [24].

CRKP infection is c onsidered a significant threat to human health worldwide owing to rapid CRKP spread, a dearth of available therapeutic options, and the major impact of these infections on patient outcomes, including lengths of hospitalization, healthcare costs, and increased mortality [1, 35]. CRKP has caused many infection control problems in healthcare systems. The results of the CRKP outcomes in this study were troubling (42.1%). In studies in Isfahan and southwestern Iran, 33.7% and 55% of K. pneumoniae isolates were CRKP positive, respectively [36, 37].

There are differences in the frequency of CRKP and antibiotic resistance across Iranian studies. These differences may result from infection control policies carried out in the hospitals studied or the indiscriminate utilization of antibiotics.

Tigecycline and colistin are the last lines of antibiotic therapy against CRKP, and resistance to these drugs has become a major clinical challenge [3]. In this investigation, tigecycline, colistin, and fosfomycin were the most effective antimicrobial agents against CRKP isolates. Tigecycline resistance was 1.6%, while in other studies, this resistance rate was 9% or 0% [5, 6].

Globally, colistin-resistant CRKP has been recorded due to the increased use of colistin [3]. In this study, 0.6% colistin resistance was observed. In other studies, resistance to colistin has been reported to be 50% and 0% [5, 6]. Although colistin is efficient in remedying infections brought on by CRKP, colistin resistance is recognized to be induced during colistin remedying and can be brought on by genetic variations and mutations in chromosomal genes [38].

In this investigation, 1.6% of CRKP isolates had triple combination resistance to tigecycline, colistin, and fosfomycin. In other studies, 9% of CRE isolates had a combination resistance to both colistin and tigecycline [5].

This is the first study in central Iran in which blaOxa−48 (46, 90.1%) and blaNDM (36, 70.5%) were the most frequently indicated carbapenemases, which is consistent with findings elsewhere [5, 6]. The proximity between Turkey (where the first blaOxa−48 gene was isolated), India (where the first blaNDM gene was isolated), and Iran, frequent travel between the countries, and ease of resistance transfer among microorganisms are probably the reasons for the high isolation of MDR and CRKP strains in blaOxa−48 and blaNDM genes from Central Iran [24].

In this research, all CRKP isolates were MDR positive, and molecular analysis determined double or triple carbapenemase gene combinations (blaOxa−48, blaNDM, and blaGES) with a co-existence of (qnrB, qnrS, and qnrA) genes.

In this research, blaNDM-positive isolates were resistant to the most antibiotics, and all of them simultaneously contained blaTEM, blaCTX−M1, blaSHV, and blaCTX−M15 resistance genes. In other studies, the simultaneous presence of these genes has also been observed [23, 39]. In our study, all of the blaOxa−48- and blaNDM-producing CRKP isolates coharbored at least one ESBL gene, which is consistent with other reports [6].

In this research, 96% of the CRKP isolates were resistant to fluoroquinolone, and the highest fluoroquinolone resistance levels were obtained by gyrA (57.1%) and qnrB (53%). In other studies, 72.7% and 62.8% of the CRKP isolates were resistant to fluoroquinolone, and the highest fluoroquinolone resistance levels were obtained by qnrS and gyrA at 86.3% and 100%, respectively [27, 40].

Resistance rates to tetracycline and fluoroquinolone in CRKP isolates differ according to the indiscriminate use of these antibiotics, infection control policies, and their geographical distribution [40].

In this research, all CRKP isolates carried the int1 gene. In other studies, int1 was found in 91.4% of isolates in Sari and 81.6% in Hamadan [24, 25]. The most common integrons in clinical settings are classes I and II [24]. These differences could be due to levels of hygiene, microbial genetic diversity, and differences in the sources of samples [24].

Conclusion

Undoubtedly, the emergence and spread of ESBL, AmpC, and carbapenemase genes in K. pneumoniae will further limit clinical therapeutic choices in Iran. Rapid and accurate diagnosis, appropriate isolation of patients with CRKP isolates, and strict, effective measures are crucial in controlling the infection and preventing the spread of these resistant isolates.