Our study demonstrated that quinolone agents were not effective against ESBL-producing K. pneumoniae isolated inpatients in Iran. ESBL-producing K. pneumoniae showed high resistant rates not only to cephalosporins but also ciprofloxacin (88%) and levofloxacin (78%). Amikacin was indicated to be effective for about 50% of the strains in our study. In Indonesia and China, ESBL-positive K. pneumoniae strains were > 90% and > 70% susceptible to amikacin, respectively [16, 17]. We also found that, among the resistant strains blaCTX−M−15 were the predominant ESBL gene. Therefore, we demonstrated that blaCTX−M−15, which is a widespread public health problem around the world, was the most common ESBL gene in ESBL-producing K. pneumoniae in Iran. The finding is in agreement with recent studies in Iran and other parts of the world [11, 17, 18]. In accordance to other studies [7, 11], our results also showed that the prevalence rates of blaTEM and blaSHV genes were high.
In the present study, a significant number of isolates (96%) carried at least one of the PMQR genes. The aac(6′)-Ib-cr gene was the most prevalent PMQR gene, in agreement with other studies[9, 19, 20]. It is suggested that aac(6′)-Ib-cr has epidemiologically strong associations with CTX-M-15 . Among the qnr genes, qnrS 20 (40%) was the most predominant, followed by qnrB 14 (28%), the occurrence of qnr alleles with aac(6′)-Ib-cr gene was in accordance with previous studies [9, 20]. Analysis of the data also revealed that the prevalence of oqxA was highest (72%) followed by oqxB (68%). Frequencies of oqxA and B genes found in this study are higher than those reported by Azargun in Tabriz, Iran in 2018 which were 33.7 for oqxA and 20.6% for oqxB . Previous studies have found that the prevalence of PMQR genes is more common in ESBL-producing K. pneumoniae [4, 9, 20]. We found that PMQR genes (aac(6′)-Ib-cr, qnrS, qnrB) were also be detected in strains containing ESBL determinants. Among the isolates containing ESBL genes, 96% were producers of PMQR. It is to be noted that ESBLs are highly prevalent in the study isolates and could have contributed to the spread of PMQRs.
In the present study MLST was used for homology analysis. ST11 international high-risk clone, the most prevalent sequence type in this collection, has been described in outbreaks of ESBL–producing K. pneumoniae in some countries such as Iran, China, Sweden and detected in a OXA-48-producing isolates from Iran and Spain [2, 11, 22, 23]. In our study, ST11 co-carried multiple resistance determinants such as blaCTX−M−15, blaTEM, blaSHV and PMQR genes including qnrB, qnrS, oqxA, oqxB and aac(6)-Ib-cr, that correlates well with earlier reports as the dominant global ESBLs are the CTX-M type beta-lactamases in K. pneumoniae [2, 22]. The presence of quinolone resistance genes aac(6′)Ib-cr and qnrB was recently reported in ST11 K. pneumoniae strains from Colombia . The second most common endemic sequence type K. pneumoniae in our study was ST893 which co-harbored both ESBLs and PMQR genes. In recent years, the emergence of ST893 have been reported from several Iranian hospitals [11, 25] which are strongly associated with the carriage of blaNDM, blaOXA−48 and ESBLs genes. Our results suggest that, ST893 is most likely endemic in Iran. ST11 and ST893 were mainly concentrated in the ICU ward, which suggests these strains may have originated in this ward and then spread to other wards in our hospital. These results suggest more attention is required in the ICU ward to avoid dissemination outbreaks of infection.
The two K. pneumoniae isolates in our study belonged to ST16 which is one of the two isolates were positive for blaCTX−M−15, blaSHV, blaTEM, aac(6′)Ib-cr and qnrS genes, whereas other isolates carried blaCTX−M−15, blaSHV and blaTEM. ST16 has been reported worldwide, showing multiple resistance determinant profiles. ST16 has been identified as a carbapenemase producer in many parts of the world and reported as an ESBL producer in Iran, Denmark and Sweden [6, 11].
Another detected ST, ST147 which is an internationally successful clone, has been reported from different parts of Iran [11, 26]. In our study, ST147 (2 isolates) has been associated with blaCTX−M−15, blaSHV, blaTEM, aac(6′)Ib-cr and oqxB. ST147 has been described in India, Greece, and Italy  and has been associated with blaVIM, blaNDM−1, blaCTX−M−15, aac(6′)-lb-cr with qnrB and armA in that country.
One isolate belonging to ST13 co-carried multiple antibiotic resistance genes such as blaCTX−M−15, blaSHV, blaTEM and aac(6′)Ib-cr. ST13 is a SLV of ST327, that has been reported to harbour blaNDM−1, blaCTX−M−15 and blaSHV in Iran . The ST392, identified in this study in one patient, ST392 was sporadically observed in different countries related to NDM-1, OXA-48 and ESBLs in Iran and to aac(6′)Ib-cr, oqxAB, blSHV, blaCTX−M−15, blaTEM−1 in Tunisia . Finally, one SHV-producing K. pneumoniae in our study belonged to ST377 and were positive for qnrS, aac(6′)Ib-cr, oqxA and qepA genes. Previously, K. pneumoniae ST377 strain carrying blaOXA−48 and ESBLs was described in Russia and Iran [11, 30]. The complexity and diversity of ESBLs and PMQR combinations detected among K. pneumoniae isolates especially successful international clones ST11 and endemic clone ST893 in this study and their potential for spread poses a real threat to the management of infections by this species in Iran.
In conclusion, this study showed high prevalence of fluoroquinolone-resistance genes in ESBL-producing K. pneumonia strains. Clonal dissemination of ESBLs carrying K. pneumoniae that co-harbour PMQR determinants have been observed. We identified the epidemically significant international and endemic STs of K. pneumoniae ST11, ST147 and ST893. Therefore, on the one hand, there is an urgent need for epidemiological and molecular studies to understand the dynamics of antibiotic resistance transmission and on the other hand, careful programs need to be implemented to prevent the spread of these strains in healthcare facilities.