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Diversity and frequency of resistance and virulence genes in blaKPC and blaNDM co-producing Klebsiella pneumoniae strains from China

Background Emergence of blaKPC and blaNDM co-producing Klebsiella pneumoniae strains have led to the limited therapeutic options for clinical treatment. Understanding the diversity and frequency of resistance and virulence genes of these isolates is of great significance. Purpose The aim of this study is to research the diversity and frequency of resistance and virulence genes in the blaKPC and blaNDM co-producing Klebsiella pneumoniae strains. Methods and Results In this study, 117 K. pneumonia strains were isolated from China, and among of which, 24 were found to be blaKPC and blaNDM co-producing with significant resistance against almost all the commonly used antibiotics. Additionally, 4 strains were hypermucoviscous and 8 showed high serum resistance. Overall, blaSHV, blaCTX-M, tetA and sul1 resistance genes found in 100% of the isolates, followed by blaTEM (95.8%), oqxA/B (91.7%), qnrB (87.5%), aac(6’)Ib-cr (83.3%), blaDHA (79.2%), rmtB (66.7%), qnrS (54.2%), cat(54.2%), floR (50.0%), sul2 (45.8%) cmlA (20.8%)andblaCMY (8.33%), respectively. What’ more, seven blaCTX-M subtypes [blaCTX-M-14 (n=18), blaCTX-M-3(n=11), blaCTX-M-65 (n=4), blaCTX-M-15 (n=3), blaCTX-M-28 (n=2), blaCTX-M-55 (n=2), blaCTX-M-22 (n=1)] and six blaSHV subtypes [blaSHV-12(n=16), blaSHV-11 (n=4), blaSHV-2a(n=1), blaSHV-1(n=1), blaSHV-38(n=1) and blaSHV-28(n=1)] were detected. The frequency of virulence genes was as follows: 100% for entB, ybtS and irp, 95.8% for mrkD, 91.66% for fimH, 79.2% for iutA, 62.5% for iroBCDE, aerobactin and kfu, 66.7% for allS, 45.8% for wcaG, 37.5% for rmpA, 20.8% for pagO and 16.7% for magA. Conclusion From this study, we concluded that the blaKPC and blaNDM co-producing Klebsiella pneumoniae strains have a high diversity and frequency of resistance and virulence genes. This study may offer hospitals important information about the control of infections caused by blaKPC and blaNDM co-producing Klebsiella pneumoniae.


Introduction
Carbapenemase-producing bacteria can hydrolyse carbapenems and most other β-lactam antibiotics which pose significant challenges to clinical diagnosis and treatment. Klebsiella pneumoniae carbapenemase (KPC) and Metallo-B-Lactamases (bla NDM ) are the two major groups of carbapenemases that produced by the most of Carbapenemase-Resistant Enterobacteriaceae strains (CRE). The bla KPC and bla NDM genes are commonly found in CRE strains in recent years. [1][2][3] Those type of the carbapenem resistance genes and other resistance genes including the key Extended-Spectrum β-lactamases (ESBLs) genes (bla CTX-M , bla SHV and bla TEM ), the fluoroquinolone resistance genes (qnrA, qnrB, qnrS, oqxA/B), aminoglycoside resistance genes (rmtA, rmtB and rmtC), chloramphenicol resistance genes (cat, floR, cmlA, cfr) and tetracycline resistance genes (tetA, tetB, tetC) are carried by the same strain and resulting in high resistance to almost all kinds of antibiotics. [4][5][6][7] The more worrisome is hypervirulent K. pneumoniae strains (hvKP) emergency sharply in recent years, especially the carbapenemase-producing hvKP related infections in immunocompromised patients which is a serious threat to the patients. [8][9][10][11] More and more researchers report that HvKP strains are characterized a number of virulence factors including aerobactin (encodes high-affinity iron chelators), rmpA (regulators of mucoid phenotype), wcaG (involved in the biosynthesis of the outer core lipopolysaccharide), allS (associated with allantoin metabolism), kfu (responsible for an iron uptake system), yptS, irp (yersiniabactin biosynthesis) and iroBCDN (salmochelin biosynthesis), entB (catecholate siderophore), fimH and mrkD (fimbrial adhesin, which mediate binding to the extracellular matrix to form the biofilm), pagO (involved in liver abscess formation by liver abscess-Kp. 9,[12][13][14][15] Understanding the diversity and frequency of resistance and virulence genes of these isolates is of great significance to disease prevention and control. For offer hospitals important information about the control of infections caused by bla KPC and bla NDM co-producing K. pneumoniae. In this study, we mainly present the diversity and frequency of resistance and virulence genes in the bla KPC and bla NDM co-producing K. pneumoniae.

Materials and methods
Isolates collection and screening of bla KPC and bla NDM genes A total of 117 non-repetitive K. pneumonia strains were isolated from sputum, cerebrospinal fluid, wound, and urine samples for routine examination between Aug. 2016 and Sept.2018 at several hospitals in Sichuan, Henan, Fujian province of China. These isolates were identified by VITEK2 Compact System (bioMérieux, France) and 16sRNA sequencing. K. pneumoniae ATCC700603 was used as the control strain for the species identification and antimicrobial susceptibility test. The bla KPC and bla NDM detection were performed according to our previous work by PCR. 9,16 Antimicrobial susceptibility testing Antimicrobial susceptibility testing of the bla KPC and bla NDM co-producing K. pneumoniae strains were performed according to the recommendations of the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). Antimicrobial agents (Oxoid, England) used used in this study included CXM (cefuroxime axetil), TZP (piperacillin-tazobactam), CAZ (ceftazidime), CRO (ceftriaxone), IPM (imipenem), MEM (meropenem), ATM (aztreonam), AMK (amikacin), CIP (ciprofloxacin), CHL (chloramphenicol), TMP-SMZ (trimethoprim/sulfamethoxazole). E. coli strain ATCC 25922 was used as quality control. 17 Hypermucoviscosity, biofilm formation and serum killing assay The hypermucoviscosity phenotype of 24 K. pneumonia was detected by string test. 18 The colonies were cultured on blood agar plate overnight at 37°, stretched by a bacteriology inoculation loop. The strain formed a viscous string of >5 mm was designated as hypermucoviscous. Biofilm formation assay was performed by crystal violet staining assay. 9 Biofilm formation in each well was measured by microplate reader (Bio-Rad, US) at optical density (OD) 595 nm. The susceptibility of the K. pneumoniae isolates to human serum was explored by an established method. 19 Briefly, K. pneumoniae strains were inoculated into LB Broth Medium and incubated at 37°C with shaking until the logarithmic phase was reached (T=4 h, OD600=0.6). 25 μL of diluted culture (containing 10 6 CFU of bacteria) and 75 μL human serum were then added into a 10×75 mm Falcon polypropylene tube and incubated at 37°C with shaking. Viable counts were checked at 0, 1, 2, and 3 h. The response to serum killing in terms of viable counts was scored on six grades as described previously method. 20

ERIC-PCR
Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) method was used to evaluate the genetic diversity of the 24 isolates, as previously described using the primers. 21 The PCR products were loaded on a 1% agarose gel with the gelred at 90 V for 40 mins, and the banding patterns were analyzed by gel imaging and analysis system. To determine the similarity rate among the acquired outcomes, Genetic diversity were analyzed using the unweighted pair-group method with arithmetic mean (UPGMA) and isolates with ≥80% similarity were treated as a single cluster. 22

Results
Antimicrobial susceptibility, hypermucoviscosity, serotyping, biofilm, serum resistance assay and ERIC-PCR typing A total of 24 bla KPC and bla NDM co-producing strains were screen from117 non-repetitive K. pneumonia strains. All the isolates were resistant to piperacillin-tazobactam, cefuroxime axetil, ceftazidime, ceftriaxone, imipenem, meropenem and aztreonam (Table 1). Among the 24 bla KPC and bla NDM co-producing strains, 16.7% (n=4) were the K1 type, while the K2, K5, K20, K57 and K54 serotype were not found ( Figure 1). String test showed that 4 (KP103L, KP48L, KP97L, KP36L) bla KPC and bla NDM co-producing K. pneumoniae isolates were hypermucoviscous. Biofilm formation was observed in all the 24 strains, with values of OD595 nm ranged from 0.33 to 2.70, whereas the mean value of the negative control wells is 0.168. Serum killing assay showed that 33.3% (n=8) of the strains were high serum resistance (Grade 5 or Grade 6). Analysis of genetic linkage among isolates by ERIC-PCR showed 34-100% similarity among 24 isolates (Table 2). Genetic diversity was established among 24 bla KPC and bla NDM co-producing K. pneumoniae isolates by detecting 15 different ERIC fingerprints with the similarity cutoff of 80% (Table 2).

Discussion
The prevalence of co-carried bla NDM and bla KPC in a single bacterial isolate in hospitals has led to heightened concerns because often makes the isolate an extremely drug-resistant variant. 2,3 In this study, 117 non-repetitive K. pneumonia strains were isolated from China, and among of which, 24 Table 1 The antibiotic resistance phenotype profile and positive rate of the resistance gene of the isolates were found to be bla KPC and bla NDM co-producing with significant resistance against almost all the commonly used antibiotics. This results showed that the positive incidence of the bla NDM and bla KPC co-producing K. pneumonia is increasing. The results were expected that all 24 isolates resist almost the all test antibiotic and biofilm formation was observed in all the 24 strains. This is a dangerous situation for antibiotic treatment because the high biofilm formation pathogenic bacteria often involved in hospital infections and always lead to the failure of antibiotic treatments. 9 Additionally, 4 strains were hypermucoviscous and 8 strains showed high serum resistance. To our knowledge, the phenotype of hypermucoviscous, biofilm formation ability and serum resistance were as the virulence evaluation criterion. 18 In recent years, more and more researchers report that the co-carried bla NDM and bla KPC K. pneumoniae strains carried a large number of resistance genes, making this isolate highly resistance against almost all the commonly used antibiotics. For example, the bla KPC-2 and bla NDM-1 co-carriage strain C. freundii 112298 existance many resistance genes including the bla SHV-12 , bla CTX-M-14 , aac (6′)-Ib-cr, bla OXA-1 , catB3, arr-3, fosA3 and sul1. 1 The bla KPC-2 and bla NDM-5 co-carriage strain ZSH6 carried    (Table 1).
Regarding the bla SHV group, bla SHV-12 (66.7%, n=16) was the most prevalent bla SHV subtype in 24 bla KPC and bla NDM co-producing strains. The threat of the high frequency and diversity of the resistance gene emergency in the bla KPC-2 and bla NDM-1 co-carriage strains should be strict surveillance and management, although its resist almost all the commonly used antibiotics. 2 Besides of the high frequency and diversity of the resistance gene, the virulence genes were also high emergency in 24 K. pneumoniae strains. In this study, we found that the frequency of virulence genes (ybtS, entB, irp, mrkD, fimH) was similar to most of others researcher's reports. However, the frequency of wcaG (45.8%), allS (66.7%) and pagO (20.8%) gene was slightly higher than our previous work. This results indicated the frequency of some virulence is rising. The high frequency of virulence factors found in these bla NDM and bla KPC bacteria is a problem for treatment. Some researchers suggested that molecular typing and virulence gene analysis are powerful tools that can shed light on Klebsiella pneumonia infections. 12,15,33,34 However, in this study, we found that some isolates were high serum resistance (Grade 5 or Grade 6) but the number of the virulence factors was less to some serum resistance strains. This results showed that how to identify the hvKP is still unknown. We suspect that the comprehensive analysis the frequency of the virulence factors, phenotype (biofilm, sting test and serum killing assay) and clinical characteristics maybe a preferable method to identitfy the hvKP strains.
In conclusion, this study demonstrated that the high frequency and diversity of the resistance and virulence factors was in the bla NDM and bla KPC co-producing K. pneumoniae making this strain resistant to almost all antibiotics. This study may offer hospitals important information about the control of infections caused by bla KPC and bla NDM co-producing Klebsiella pneumoniae.

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