The emergence of the hypervirulent Klebsiella pneumoniae (hvKp) strains among circulating clonal complex 147 (CC147) harbouring blaNDM/OXA-48 carbapenemases in a tertiary care center of Iran

Background Klebsiella pneumoniae is a public health concern because of its ability to develop multidrug resistance and hypervirulent genotypes, of those capsular types K1 and K2 cause community and nosocomial life-threatening infections. This study aimed to determine the antibiotic susceptibility patterns and genotypic traits of a collection of Klebsiella spp. isolates. Furthermore, the clonal relatedness of blaNDM producing strains was investigated. Methods During a 19-months surveillance study, 122 Klebsiella spp. isolates were cultured from extraintestinal specimens of patients admitted to the tertiary referral hospital in Semnan, Iran. Isolates were identified using biochemical tests and subjected to determination of phylogroups, capsular types and virulence/resistance genes content. Hypervirulent K. pneumoniae (hvKp) strains were detected genotypically, and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting was used to determine the clonality of blaNDM producing strains. Results Multidrug resistant phenotype was detected in 75 (61.5%) isolates and amikacin was found as the most potent antibiotic with the susceptibility rate of 85.2%. The carbapenemase genes were detected in 45 (36.8%) strains, including 21 (17.2%) blaOXA-48, 7 (5.6%) blaNDM-1, 14 (11.4%) blaNDM-1/OXA-48 and 3 (2.4%) blaIMP- carrying strains, while 55 (45.08%) isolates showed carbapenem resistant phenotype. The first blaNDM-1 carrying strain was cultured from a sputum specimen on March 2015, while the last positive one was recovered from blood culture on September 2016. Most of the isolates (80.3%) belonged to phylogroup I, and blaNDM-1 was identified among all three phylogroups. The ERIC-PCR clustered the 101 blaNDM negative and 21 blaNDM-1 positive isolates into 25 and five clusters, respectively, and the latter group belonged to clonal complex 147 (CC147). One K1 and 15 K2 blaNDM-1 negative isolates were detected, of those three strains were identified as hvKp. Five K2 positive strains, including four blaOXA-48 producer and one hvKp sequence type 86 (ST86) were carbapenem resistant. Among carbapenem resistant isolates, CC147 strains harboured higher rates of siderophores iutA and ybtS. Conclusion The present findings showed a hospital circulation of CC147 blaNDM-1 or blaNDM-1/OXA-48 producing strains, disseminated in different wards. The hvKp/ST86 strain expressing K2 capsular type and carbapenem resistant phenotype wasn’t reported from Iran so far. So, it seems that we must be aware of the emergence and spread of new K. pneumoniae clones associated with resistant and hypermucoviscous phenotypes.


Background
Klebsiella pneumoniae, one of the most important members of the Enterobacteriaceae family, is the leading cause of both of the community and healthcare-associated infections. The capsule, as a critical virulence factor in this organism, plays an important role in its pathogenesis and avoids phagocytosis [1]. Over the past decade, the emergence of hypervirulent variants of K. pneumoniae (hvKp) which characteristically express hypermucoviscosity phenotype caused serious concerns [2]. These strains carry virulence genes associated with invasive disease and may cause severe infections such as pyogenic liver abscesses and endophthalmitis, in immunocompetent, healthy individuals [2]. While the hvKp strains were rarely resistant to commonly used antibiotics when firstly described, the emergence of multidrug resistant (MDR) phenotypes among these strains are increasingly reported in recent years due to the dissemination of mobile genetic elements encoding drug resistance [2]. Of special concern is the acquisition of carbapenem resistance genes and development of carbapenem resistant phenotype, since these agents are the last resort of antibiotics for treatment of infections caused by multidrug resistant organisms [2].
Increasing recognition of carbapenemase producing K. pneumoniae, including class A (KPC), class B (IMP, VIM and NDM) and class D (OXA-48-like enzymes) carbapenemases has led to international concern, as they are carried on transposable elements in association with other resistance determinants, such as Extended spectrum β-lactamases (ESBLs), ampC cephalosporinases and 16S rRNA methyltransferases [3]. Indeed, the convergence of carbapenemase production and hypermucoviscosity phenotype in this organism poses an important threat to public health.
The bla NDM , a relatively newly described Metallo-βlactamase (MBL), was first identified in K. pneumoniae and E. coli isolated from a Swedish patient who was hospitalized in India in 2008 [4]. Since then, it has spread worldwide and now NDM producing Gram-negative bacilli have been reported from more than 40 countries, and the Indian subcontinent and the Middle East are considered as the main reservoirs for bla NDM producing bacteria [5]. Iran, as one of the Middle East countries, neighbor countries where the NDM and OXA-48 producing bacteria are endemic [6]. While bla NDM producing K. pneumoniae isolates have been reported from different cities of Iran in recent years, sequence types of these isolates are determined and published from three cities located in the center, south, and south-east of this country [6][7][8]. In our previous study, we reported a relatively high prevalence of bla OXA-48 /bla NDM-1 producing Enterobacteriaceae isolates collected from the large tertiary hospital of Semnan [9], an important city along the historical Silk Road. So in this survey, our goal was first to determine the phylogenetic groups, capsular genotypes, hypermucoviscosity biomarkers, and resistance determinants of K. pneumoniae isolates collected during 19-months surveillance study and second, to carry out sequence typing of representatives of NDM producing isolates based on the Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting.

Sample collection
A 19-months surveillance study was conducted in the main tertiary teaching hospital of Semnan, Iran (Kosar hospital). During March 2015 to September 2016, 122 non-duplicate K. pneumoniae isolates were recovered from clinical specimens of patients admitted to the hospital. The isolates were cultured from different extraintestinal specimens including urine, wound, sputum, blood and tracheal aspirate. Specimens were collected as the routine diagnostic purposes. K. pneumoniae isolates were identified based on the biochemical reactions, including reaction on Triple Sugar Iron (TSI) agar, SH2/Indole/ Motility (SIM) pattern, growth on Simmon-citrate agar medium, urease production on urea agar, Methyl Red/ Vogues Proskauer (MR/VP), and Ornithine decarboxylase (OD) test. Isolates were confirmed by PCR in which both k. pneumoniae subsp. Pneumoniae and subsp. ozaenae give a 130 bp band, and subsp. rhinoscleromatis is negative [10]. K. oxytoca species were identified based on the VP +/Indole +/OD negative, tests results [11].

Phylogenetic determination and clonal relatedness
For phylogenetic analysis, gyrA PCR-RFLP using restriction enzymes TaqI and HaeIII was performed as described by Brisse et al. [21]. The clonality of strains was determined by ERIC-PCR fingerprinting and the obtained dendograms were analyzed with Bionumerics software, version 6.1 (Applied Maths, Sint-Martens-Laten, Belgium). The similarities in amplicon profiles were compared using a Dice coefficient at 1% tolerance and 0.5% optimization, and a dendogram was constructed using the unweighted-pair group method, with a cutoff of 80% similarity [22]. Sequence type of isolates was determined for representatives of NDM positive strains of each cluster of ERIC dendogram according to the K. pneumoniae MLST website (https ://bigsd b.paste ur.fr/klebs iella /klebs iella .html).

Statistical analysis
Dichotomous variables were described using frequencies and percentages, and they were compared using Chi square test, as appropriate. The criterion for statistical significance was P < 0.05. Data were analyzed with software SPSS version 16.

Genetic relatedness and sequence typing of bla NDM producers
Based on the ERIC-fingerprinting of bla NDM negative isolates, 25 clusters were detected, including of two to 10 isolates in each cluster. Twenty-six isolates were also found as singletons (Fig. 2). In contrast, NDM carrying strains were grouped into five clusters (Fig. 3). Consequently, as the representatives of NDM producers, one strain was selected from each of the cluster and subjected to MLST assay. Two sequence types, ST392 (n = 9) and ST147 (n = 12) were identified among NDM positive isolates. Noted that the ST392 is the clonal complex (CC) of ST147. The first bla NDM-1 strain was obtained on March 2015 from internal intensive care unit (ICU) followed by the couple of isolates retrieved on May from surgical ICU. On June, there was only one strain isolated from a patient hospitalized in internal ward. On July, we obtained one bla NDM-1 isolate from each of the internal ICU and surgical ICU sites. On August, there were three isolates; two from internal ICU and one from internal ward. On the following months from October 2015 to February 2016, nine strains were isolated from patients hospitalized at internal ICU. On March, we obtained one isolate from internal ward and afterward we received two other isolates from coronary care unit (CCU) and surgery wards on April. The last positive strain recovered from a dialysis patient whom was initially hospitalized in internal ICU on September 2016.

Phylogroups, capsular genotypes and hypervirulent clones
Except for Klebsiella oxytoca strain (strain no. 511b) which showed a different RFLP-pattern, all the remaining 121 studied isolates were grouped in three phylogroups, including 98 (80.3%) in group I, 20 (16.4%) in group II and three (2.5%) in group III. Most of the NDM-1 producing isolates, including 17 (81%) strains, belonged to the group I, and the remaining four strains belonged to group II and III. Of the CTX-M genes, the CTX-M-G2,  isolates were identified as hvKp. Virulence genes iutA, mrkD and ybtS were detected in these three hvKp strains, except for K1 isolate which was negative for the latter gene. The two K2 + /hvkp strains (strains 533 and 290) were identified as ST86 using the hvKP-K2 multiplex PCR. As expected, this multiplex PCR was completely negative for the only K2 + /phylogroup II strain (strain 608). The alls gene was not detected among study isolates. None of the bla NDM-1 producers were positive for K1/K2 capsular types and were not identified as hvkp.

Discussion
The first report of NDM producing Gram-negative bacilli from Iran was described in 2013 in a K. pneumoniae strain, which was cultured from the urine specimen of a patient with kidney transplant rejection history [23].
In the current study, we investigated the molecular epidemiology of a group of bla NDM-1 producing isolates cultured from various extraintestinal specimens collected from Kosar hospital, Semnan, Iran. In our study, NDM-1 producing K. pneumoniae strains were mostly derived from urine specimens, followed by sputum of patients admitted to different hospital wards. In our study and comparison to NDM negative isolates, bla NDM-1 carrying strains were much resistant to different classes of β-lactam and non β-lactam antibiotics by disk diffusion assay. It has been shown that tigecycline, fosfomycin and colistin are the most potent agents against NDM producing isolates [8]. While we didn't determine the susceptibility patterns of NDM producing strains against these antibiotics, amikacin which found with the highest susceptibility rates may be considered as a treatment option due to some limitation of the extensive clinical usage of the three aforementioned antibiotics.
Phylogrouping of study Klebsiella isolates identified three groups, with the phylogroup I as the dominant group, followed by group II and III. It has been shown that the level of resistance for most of the antibiotics is highest among phylogroup I, intermediate in group II and lowest in group III, with the highest number of normal microbiota among the latter group [24]. Accordingly, most of the bla NDM-1 producing strains (81%) belonged to group I and co-harbored different resistance genes, including bla SHV-, CTX-M-G1, qnrS, aac6-Ib-cr and aac3IIa. While the resistance is a critical parameter for the transmission of phylogroup I K. pneumoniae strains, detection of NDM/OXA-48 carbapenemases and consequently carbapenem resistant phenotype among phylogroup II (strains: 500A, 718, 293 M) and III (isolate: 247) strains, confirm the role of this mainly nosocomial and opportunistic pathogen in the dissemination of resistance elements.
Among the bla NDM-1 producers, 11 (52.3%) strains coharboured any β-lactamase gene, CTX-M; any PMQR; and 16S rRNA methylase gene armA or rmtC. PMQR coexisting with any β-lactamase gene was also high (21 [100%]). A strong association between the carriage of NDM and 16S rRNA methylase encoding gene, specifically rmtC methylase has been shown [25]. While the prevalence of rmtC among bla NDM-1 producing strains was higher than the negative ones, this association was found borderline. In contrast, the co-occurrence of armA or the other aminoglycoside resistance genes with NDM was found significant. So our results indicate that NDM producing Klebsiella isolates, have acquired a broad spectrum of singular and different resistance determinants.
It has been reported that carbapenemase producing K. pneumoniae strains have a different population and less genetic diversity as compared to carbapenem susceptible isolates [26]. In our study, ERIC-PCR showed five clusters and higher genetic homogeneity among bla NDM-1 producers as compared to negative ones. Accordingly, the MLST assay identified two STs among NDM producing isolates. ST392, which is an SLV of ST147, has been associated with the carriage of bla KPC , bla NDM and bla OXA-48 [27]. In the current study, ST392 has detected among NDM positive isolates with different ERIC patterns. Another detected ST, ST147 which is an international clone, has been reported from center, south and south east of Iran [6][7][8]. In our study, ST147 has been associated with CTX-M-G1, bla SHV-, aac6Ib-cr and armA. This ST has been recognized as a pandemic clone and is considered as a threat to public health worldwide [28]. ST147 was firstly observed by Papagiannitsis et al., as hosting the bla VIM gene, and then Samuelson et al. reported this clone among K. pneumoniae isolates imported to Scandinavia, mostly from Greece [28]. So, these findings imply that ST147 has the potential to acquire different resistance elements, of note, bla NDM/OXA-48 carbapenemases and facilitate their rapid spread into the other pandemic clones of K. pneumoniae.
String test, a phenotypic assay that is commonly used to identify the hvKp strains, has been shown to performed suboptimally, particularly in low prevalence areas [18]. So, the identification of some genetic markers including peg344, irob, rmpA, rmpA2 and iucA has been suggested for accurate differentiation between hvKp and classical K. pneumoniae strains [18]. Accordingly, one K1 and two K2 ST86 strains were identified as hvKp, of those one ST86 strain was MDR and carbapenem resistant. While these capsular types have not generally been associated with acquired resistance genes at the time which were identified, in the last few years increasing reports of resistant strains were observed among these genotypes [29]. In agreement with these new reports, four out of five carbapenem resistant K2 strains harboured the bla OXA-48 gene. It has been reported by Turton et al., that isolates of CC147 carrying bla OXA-48 or bla NDM which was resistant to colistin, harboured many antibiotic resistance determinants, and contained a quarter of the virulence genes which were found in the K1-ST23/OXA-48 + isolates [30]. While our CC147 strains (all carrying NDM) were K1/K2/hvkp negative, they harboured relatively higher rates of siderophores iutA and ybtS as compared to another carbapenem resistant NDM negative isolates. So, concerning this finding that the acquisition of any one of the siderophore clusters increases the risk of complicated infections [2,30], the combination of virulence and antibiotic resistance in this pandemic clone is extremely worrying.
Our study had some limitations. The bla OXA-48 producers and K1/K2 strains were not subjected to sequence type determination. Furthermore, the TEMand SHV-variants of positive isolates were not determined. We investigated the carriage of some limited virulence factors among carbapenem resistant strains, while the other important virulence determinants remained to be characterized among both of carbapenem susceptible and resistant isolates.

Conclusions
In summary, clonal dissemination of bla NDM-1 carrying K. pneumoniae that co-harbour different β-lactamases, aminoglycoside modifying enzymes, and PMQR determinants have been observed. Isolation of carbapenem resistant K. pneumoniae strains from clinical sources has been reported from Iran, previously. However, their association with K1 or K2 hypervirulent capsular types has never been reported. The emergence of the CC147 carbapenem resistant K. pneumoniae strains warrants urgent surveillance because not only are they considered as international clone, but also they simultaneously have higher rates of siderophores in a pandrug resistance profile.