Development and evaluation of a LAMP assay for differentiating Carbapenem-Resistant Acinetobacter baumannii clinical strains harboring blaOXA-23

Background Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen in hospital-acquired infections, and the resistance to carbapenems has been observed increasingly worldwide. Oxacillinase produced by blaOXA-23 is one of the predominant carbapenem resistance mechanisms in A. baumannii, which is highly prevalent worldwide, especially in China. The rapid identification of blaOXA-23 may give a valuable hint for the administration of directed antimicrobial therapy. Method In this study, we aimed to develop a LAMP-based detection for the blaOXA-23 gene; clinical samples of A. baumannii were used to determine the sensitivity and specificity of this method compared to phenotypic antimicrobial susceptibility testing and traditional PCR method. MLST was performed to investigate the epidemiology of A. baumannii bacterial population. Results Compared to the antimicrobial susceptibility testing, the sensitivity and specificity of LAMP in detecting blaOXA-23 was 88.4% and 97.7%, respectively. However, the LAMP method was found to be much simpler and the result could be available in a shorter period (within 60 minutes) when compared to conventional PCR and phenotypic susceptibility testing. The 113 isolates could be clustered into 30 sequence types (STs), and majority (83/113) of these strains belong to clonal complex 92 (CC92), which is also the dominant CC in the China. isolates, and the assembled sequences were aligned by using BLAST to assign the allelic numbers and sequence types (STs). Then the results were compared with the available alleles in the A. baumannii MLST (Oxford) database. eBURST analysis (http://eBURST.mlst.net/) was further conducted to investigate the genetic relationships and clonal complexes (CCs) of these isolates.

associated pneumonia, urinary tract infections, pneumonia and bacteraemia [ 1]. This bacterium has a particular predilection for immunocompromised patients and severely ill patients in Intensive Care Units (ICUs), and is associated with substantial morbidity and mortality rates and high-cost health care [ 2].
One reason for the emergent interest in A. baumannii due to its great capability of acquiring new mechanisms of resistance adapt to any environment and eventually exhibiting multidrug resistance, which have been of significant medical concern [ 3]. Carbapenems represent the most versatile family of β-lactamases, and are recommended as last choice options for the treatment of serious infections. However, with the overuse of carbapenems, the carbapenem-resistant A. baumannii (CRAB) isolates have been reported frequently in recent years, and the resistance rate has increased to about 60% or even higher [ 4,5]. Although the resistance mechanisms of A. baumannii to antibiotics are complicated, the acquisition of β-lactamases with carbapenem hydrolyzing activity has been attributed mainly to the resistance against these agents [ 1]. Among the most widely spread carbapenem-resistant oxacillinase (prefix OXA) βlactamases genes, the plasmid-mediated bla OXA-23 is the predominant oxacillinase responsible for the majority of phenotypic resistance to carbapenems in A. baumannii detected around the world [ [6][7][8][9][10]. The production of a low-copy-number plasmid vector carrying bla OXA-23 gene by an A. baumannii strain is enough to confer resistance to the carbapenems [ 11]. Therefore, the bla OXA-23 gene could be potentially considered as a surrogate marker for detecting the dissemination of CRAB strains carrying the bla OXA-23 in clinic.
Rapid detection of pathogens and their resistance characterization is of suggestive significance, since it may lead to administration of directed antimicrobial therapy at an early stage, reduce the unnecessary earlier use of broad-spectrum agents, potentially result in better outcomes, and less was prepared by serial 10-fold dilutions to yield concentrations ranging from 324 ng/μl to 0.03 pg/μl.

Primer Design for LAMP Assay
To design bla OXA-23 specific LAMP primers, the sequence of bla OXA-23 with accession number   [ 15]. The seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were amplified for all isolates, and the assembled sequences were aligned by using BLAST to assign the allelic numbers and sequence types (STs). Then the results were compared with the available alleles in the A. baumannii MLST (Oxford) database. eBURST analysis (http://eBURST.mlst.net/) was further conducted to investigate the genetic relationships and clonal complexes (CCs) of these isolates.
Statistics SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) was applied for the statistical analysis. The Pearson Chi-Squared test was performed to compare the consistency between LAMP based method VITEK 2 system and a conventional PCR assay.

Results
LAMP reactions for bla  LAMP reactions were carried out according to the manufacturer's instructions, and all the procedures were standardized at 65•C for 60 min. To select the optimal primers for the bla OXA-23 , the turbidity curves of the four sets of designed primers under the same condition with synthesized bla OXA-23 DNA templates were observed. All the primer sets enabled successful amplification, of which the bla OXA-23 -1 primer set (Table 1) amplified the target sequence within the shortest time as shown in Figure 1, therefore, was chosen as the optimal primer set.  Amplification was performed at 65•C for 60min, and the turbidity was monitored with a Loopamp realtime turbidimeter at a 650nm -absorbance every 6s.
The detection limit of LAMP reaction was performed using 10-fold serial diluted bla OXA-23 DNA from 324 ng/μl to 0.032 pg/μl. As shown in Figure 3, the detection limits of the real-time turbidity was about 32.4 pg/μl. The Kappa values between bla OXA-23-LAMP assay and VITEK 2 system was 0.837, between bla OXA-23-LAMP assay and PCR was 0.982, respectively. The consistency of these assays was quite satisfied. However, the procedure of the LAMP assay was less complicated when compared to the PCR, more rapid (available within 60 minutes) compared with the VITEK 2 results (18-24 hours). The results of LAMP assay and conventional PCR was highly consistent in identification of bla OXA-23 in this research, both of which could potentially serve as a surrogate marker for the presence of CRAB carrying the bla OXA-23 .    31], and even next generation sequencing (NGS) [ 32] were used to establish the rapid detection assays.
In this research, we described a LAMP-based diagnosis assay for the rapid detection of CRAB harboring the most prevalent carbapenemase gene bla OXA-23 . Sample for this assay was cultureindependent, and genomic DNA extracted from sputum samples by commercial kits or just by direct boiling method greatly shorts the preparing work. When applied the LAMP assay, a positive reaction could be completed within an hour. 113 clinical samples of A. baumannii were used to determine the sensitivity and specificity of this method, and by comparing to phenotypic susceptibility testing, the sensitivity and specificity of LAMP in detecting bla OXA-23 was 88.4% and 97.7%, respectively. Eight phenotypic carbapenem-resistance strains were bla OXA-23 -negative in both LAMP assay and PCR. A. baumannii resistant is highly complex, it is possible that other mechanisms, such as the production of other carbapenemases, hyperexpression of efflux pump, changes in outer membrane proteins (OMPs) were attributed to the carbapenem-resistance in these eight isolates. One carbapenem susceptible A.
baumannii strain was presented bla OXA-23 -positive result in the LAMP assay. Very likely it was a falsepositive result, however, it is possible that the prodution of the carbapenemases/ the copy number of bla OXA-23 gene was too low to be detected by the avilable phenotypic susceptibility testing/ the conventional PCR.
It has been demonstrated that the epidemiology of the A. baumannii bacterial population closely related to their antimicrobial resistances. The great ability of acquiring resistance determinants and propensity to adapt to any environment resulted by their extraordinary genomic plasticity and powerful adaptability has led to the simultaneous presence of dominant A. baumannii clonal lineages, such as international clones I and II, and increasing prevalence in international hospitals all over the world [ 33]. A need to monitor the epidemiology of A. baumannii and its associated antimicrobial resistances is reinforced. So, MLST was employed to investigate the epidemiology of A. baumannii bacterial population. The 113 isolates could be clustered into 30 sequence types (STs), and majority (83/113) of these strains belong to clonal complex 92 (CC92), which was corresponded to international clonal lineage II. CC92 is the dominant CC in the Chinese mainland [ Availability of data and material All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.   Figure 1 Four sets of primers were used to amplify blaOXA-23 genes under the same conditions (at 65•C for 60min). The assay was monitored for the product of LAMP reaction, magnesium pyrophosphate, at optical density 650nm every 6s. The blaOXA-23-1 primer set was chosen as the most appropriate primers for the rapid detection of blaOXA-23.

Figure 2
Effect of differing temperatures on the efficiency of detection of blaOXA-23 by LAMP assay.
Amplification was performed at 65•C for 60min, and the turbidity was monitored with a Loopamp real-time turbidimeter at a 650nm -absorbance every 6s.