High Prevalence of 16S rRNA methylase genes High Prevalence of 16S rRNA methylase genes Among Carbapenem-resistant Hypervirulent Klebsiella pneumoniae Isolates in China

Background : the existence of 16S rRNA methylase genes would increase treatment difficulty of patients infected with CR-hvKP strains, this study was aimed to testify the prevalence of the 16S rRNA methylase genes genes in the CR-hvKP strains in China.Methods : Thirty-nine carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates collected from a Chinese hospital during the whole year of 2018 were evaluated to characterize the prevalence of 16S rRNA methylase genes. Results : In tatal 66.7% (26/39) of the CR-hvKP isolates were found to carry 16S rRNA methylase genes, and the most frequently detected gene was armA (11,42.3%), followed by rmtB (8,30.8%),and 7 CR-hvKP strains were found to carry both armA and rmtB (26.9%). All the clinical isolates were found to carry at least one carbapenemase gene,with KPC-2 (79.5%,31/39), NDM-1 (10.3%,4/39), and cocarrying KPC-2 and NDM-1 (10.3%,4/39). A total of 89.7% (35/39) isolates carried ESBL genes, including 61.5% (24/39) blaSHV-1 ,71.8% (28/39) blaTEM-1 and 89.7% (35/39) blaCTX-M-1 4. All except four isolates (89.7%,35/39) harbored PMQR genes,with qnrS (82.1%,32/39), aac(6’)-Ib-cr (79.5%,31/39), qnrB (2.6%,1/39).All the 16S rRNA methylase genes-positive CR-hvKP strains were firstly found to cocarry carbapenemase genes, ESBL genes and PMQR genes simultaneously. The most prevalent virulence genes were rmpA2 and entB (100%, 39/39),followed genes including aerobact , repA , kfuBC and wcaG were highly clustered in the K1/K2 16S rRNA methylase genes-positive CR-hvKP strains compared to non-K1/K2 16S rRNA methylase genes-positive strains. Our data showed that


Background
Klebsiella pneumoniae is one of the common pathogens of nosocomial infections including bloodstream infections, pneumonia, urinary tract infections and liver abscesses [1].Recently carbapenem-resistant hypervirulent klebsiella pneumoniae (CR-hvKP) infections have been reported widely in China [2][3][4].Due to acquisition of the carbapenem-resistance plasmid by the hvKP strains or the acquisition of the virulence plasmid by the CRKP strains, CR-hvKP strains simultaneously exhibit the features of hyperresistance, hypervirulence, and high transmissibility so that they should be regarded as a real superbug which need to raise enough concerns [5].
In spite of ototoxicity and nephrotoxicity, aminoglycosides (AGs) are few optional semisynthetic antimicrobial agents exhibiting high-susceptibility and excellent postantibiotic effect against CRKP strains [6,7].The biological mechanisms of resistance to aminoglycosides include decreased permeability, increased efflux, enzymatic modification, and modifications of the 30S ribosomal subunit that interferes with binding of the aminoglycosides [8]. Over the past few decades some studies have found alarmingly high rate of 16S rRNA methylase genes among CRKP strains [9,10], it precludes the use of key aminoglycosides (gentamicin, tobramycin, and amikacin) even when carbapenems have already been excluded from the treatment option. Obvious the existence of 16S rRNA methylase genes would increase treatment difficulty of patients infected with CR-hvKP strains.Thus, this study was aimed to testify the prevalence of the 16S rRNA methylase genes genes in the CR-hvKP strains in China. guidelines [12].Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as the quality control. E. coli J53 was used in the conjugation experiments.

Conjugation experiments
Luria-Bertani (LB) mating experiments were performed using sodium azide-resistant E. coli J53 as the recipient to determine whether the 16S rRNA methylase genes or the carbapenemase genes are transferable.Transconjugants were selected on LB plates containing sodium azide (100 mg/L) plus gentamicin (30 mg/L) or imipenem (4 mg/L), and finally confirmed by PCR and pulsed field gel electrophoresis (PFGE) [19]. We also performed PCR to confirm whether the transconjugants also contained the other antibioticresistance genes that were found in the donor strains.

Results
The clinical characteristics and the association of antibiotic susceptibilities with

16S rRNA methylase genes in CR-hvKP isolates
Overall, 39 patients had proven or suspected acquisition of CP-hvKP, more than half of all patients (64.1%) had the carbapenems application empirically, but only 7 patients (17.9%) had the aminoglycosides application. On the basis of the presence of 16S rRNA methylase genes in these CR-hvKP strains, all CR-hvKP strains were divided into two groups (16S rRNA methylase genes-positive strains and 16S rRNA methylase genes-negative strains). isolates ( p < 0.05).

Molecular characteristics
The PFGE-based fingerprints of the CR-hvKP isolates displayed three different clusters (named A-C) using a similarity cutoff value of 80% (Fig. 1

Conjugation experiments
In conjugation experiments, twenty-six 16S rRNA methylase genes positive strains were transconjugants.

Discussion
Over the past few decades, hvKP has globally emerged,causing invasive infections since the first clinical hvKP report in 1986 [21]. Although initial isolates of hvKp were antimicrobial sensitive,clonal complexes of hypervirulent (hvKP) and multidrug-resistant (MDR) strains are non-overlapping [22].In this study we collected 39 CR-hvKP strains, including 31 ST11 CRKP carrying the pLVPK like plasmid ,4 ST23, 2 ST65 and 2 ST86 hvKP carrying the carbapenemase plasmid.It was consistant with the evolution hypothesis of MDR-hvKP occurring by two mechanisms [21]. The first and more frequent was via XDR cKp acquiring a modified hvKp virulence plasmid, and the converse was via hvKp strains gaining antimicrobial resistance genes by acquisition of resistance plasmids or by the insertion of resistance elements into hvKp's virulence plasmid [5,21]. To date,though such CR-hvKP strains have been described only in China; the prospect of CR-hvKp undergoing wider dissemination is concerning.
Because of the expensive cost, the application of tigecycline and polymyxin in clinical treatment were severely limited.Considering that CRKP isolates were generally susceptible to aminoglycosides, these drugs were widely used for treating CRKP infections [6] in spite of ototoxicity and nephrotoxicity.However, increased manifestations of antimicrobial resistance mediated by plasmids, especially 16S rRNA methylase, and carbapenemase in MDR K. pneumoniae, had been observed in China [23]. In this study the 16S rRNA methylase genes were highly prevalent in CR-hvKP isolates, The most frequently detected 16S rRNA methylase genes was armA, followed by rmtB and both two in all isolates. As described in a previous study [24] [4]. In addition, ST23, ST65 and ST86 were also found to be associated with 16S rRNA methylase genespositive CR-hvKP.
Several potential limitations and caveats of this study are noteworthy, including its retrospective nature and a relatively small study population. In this study, 39 isolates used in the analysis were unable to clearly elucidate the 16S rRNA methylase genespositive CR-hvKP epidemiology.Therefore, there may be selection bias, which limits the general application of study results to other areas.

Conclusions
In conclusion, 16S rRNA methylase genes are highly prevalent in CR-hvKP clinical isolates in our hospital, and 16S rRNA methylase genes-positve CR-hvKP strains especially for ST11 will be difficult to eliminate and control because of both horizontal transfer and clonal spread.Furthermore, the 16S rRNA methylase genes could be cotransferred with blaKPC, suggesting that CR-hvKP could acquire transferable resistance elements as independent events from an external source, of which we should keep alert Abbreviations

Ethics approval and consent to participate
The study has been evaluated by the Ethics Committee of the First Affiliated Hospital of Nanchang University. Patients involved in the study were anonymized, no informed consent was acquired because of the retrospective study.

Consent for publication
Not applicable

Availability of data and material
The data that support the findings of this study are available from the corresponding author upon reasonable request

Competing interests
The author reports no conflicts of interest in this work.

Funding
Financial support was provided by the National Natural Science Foundation of China (81860368).

Author contributions
FLD and DL performed the laboratory measurements. YL and WJL made substantial contributions to conception and design. WZ and YL revised the manuscript critically for important intellectual content. DL and DDW participated in experimental design and data analysis. WJL drafted the manuscript. All authors read and approved the final manuscript.