Extensively drug-resistant Raoultella planticola carrying multiple resistance genes including blaNDM-1

Case presentation: In this study, an XDR R. planticola strain, RP01, was isolated from a Chinese patient with multiple chronic diseases. Antimicrobial susceptibility testing showed that RP01 was resistant to almost all clinically available antibiotics except tigecycline. PCR and sequencing revealed the presence of blaNDM-1, encoding a metallo-b-lactamase with hydrolysing activity against carbapenems. Further genomic sequencing and ResFinder analysis identified 20 resistance genes, encoding resistance to b-lactams, aminoglycosides, sulfonamides, tetracycline, fluoroquinolones, trimethoprim and fosfomycin.


Introduction
Raoultella planticola is a Gram-negative anaerobic oxidasenegative non-motile bacillus belonging to family Enterobacteriaceae (Drancourt et al., 2001).This bacterium used to be considered an environmental organism residing in water and soil.However, an increasing number of cases of R. planticola infection, including bacteraemia (Hu et al., 2012), soft tissue infection (O'Connell et al., 2010), pancreatitis (Alves et al., 2007) and urinary tract infection (Olson et al., 2012), have recently been reported.Gastrointestinal and oropharyngeal colonization of the bacterium has also been described in adults and newborns (Podschun et al., 1998;Toivanen et al., 1999).Although bla KPC -producing strains with a multi-drug resistance phenotype have been reported (Castanheira et al., 2009), most R. planticola isolates were sensitive to cephalosporins, aminoglycosides, fluoroquinolones and carbapenems.In this study, we report the first extensively drug-resistant (XDR) R. planticola clinical isolate expressing the bla NDM-1 gene, with resistance to almost all clinically available antibiotics except tigecycline and colistin; the resistance gene profiles were analysed by high throughput sequencing and ResFinder analysis.

Case report
On 26 May 2012, an 82-year-old male patient was admitted to the First Affiliated Hospital of Guangzhou Medical University (FAHG) in Guangzhou, China, because of cough and shortness of breath for 10 days.He had also suffered chronic obstructive pulmonary disease, coronary disease and hypertension.A sputum sample from the patient was cultured and grew a Gram-negative bacillus with a bacterial count of 10 7 c.f.u.ml 21 .By using the Vitek2 Biochemical Identification system, the isolate was identified as R. planticola with a 99 % probability.PCR and DNA sequencing of the 16S rRNA gene confirmed the biochemical identification results.The isolate was designated RP01 and antimicrobial susceptibility testing was performed by Vitek2 Automated System and Etest (bioMe ´rieux).The results were interpreted according to the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2012).RP01 exhibited an XDR phenotype.It was resistant to all b-lactams, aminoglycosides, sulfonamides, tetracycline, fluoroquinolones, trimethoprim and fosfomycin (Table 1).The isolate was susceptible to tigecycline and colistin with MIC values of 2 and 0.125 mg ml 21 , respectively.After comprehensive analysis of other examination results, including normal body temperature, normal white blood cell count, and slightly elevated levels of serum procalcitonin, the responsible physicians felt the clinical scenario was more consistent with colonization rather than infection.The patient received symptomatic treatment for 2 weeks, which alleviated his respiratory distress.Further specific treatment for coronary disease was then carried out and the patient was discharged after 32 days.

Investigations
Although the modified Hodge test for RP01 showed a negative result, it was positive by the imipenem/EDTA double-disc synergy test, indicating the presence of metallo-b-lactamase genes.Multiplex PCR detection for 11 acquired carbapenemase genes was carried out as described previously (Poirel et al., 2011) and identified the bla NDM-1 gene in RP01.The PCR product was further sequenced to confirm the bla NDM-1 gene sequence.RP01 was the first clinical isolate producing bla NDM-1 identified in FAHG, and no bla NDM-1 -positive strains were detected from other hospitalized patients during the patient's stay.
Attempts to transfer the bla NDM-1 gene to donor strain E. coli J53 by conjugation failed.To investigate the molecular mechanism for the XDR phenotype, high-throughput DNA sequencing was carried out by the Ion Torrent personal genome machine (Life Technologies).Total DNA of RP01 was prepared by DNA extraction kits (Qiagen) and sequenced following the manufacturer's protocols.A total of 3 294 078 reads with a mean length of 199 bp were obtained, which represented about 796 coverage data for the RP01 genome.De novo assembly was performed using the MIRA assembler (version 3.4.0.1) and 170 large contigs (.500 bp) were obtained.Antibiotic-resistant genes were analysed in the ResFinder database (Zankari et al., 2012) using the assembled contigs.A total of 20 resistance genes were identified on different contigs, including 5 for b-lactam resistance (blaCTX-M-3, NDM-1, OXA-30, SHV-12 and TEM-122), 4 for aminoglycoside resistance [aac(6')Ib-cr, aadA16, aadA5 and armA], 3 for macrolide resistance [mph(A), mph(E) and msr(E)], 2 for quinolone resistance [aac(6')Ib-cr and qnrB6], 2 for trimethoprim resistance (dfrA1 and dfrA27), 1 for fosfomycin resistance (fosA), 1 for phenicol resistance (catB3), 1 for rifampicin resistance (ARR-3), 1 for sulfonamide resistance (sul1) and 1 for tetracycline resistance (tetD).The presence of aac(6')Ib-cr contributes to both aminoglycoside and quinolone resistance (Strahilevitz et al., 2009).The bla NDM-1 gene was located on a 5.8 kb contig with 100 % sequence homology to the counterparts of pNDM-HN380, an IncX3 plasmid isolated from Enterobacteriaceae strains carrying bla NDM-1 in China (Ho et al., 2012).Alignment of pNDM-HN380 sequence to the sequencing reads identified all functional regions for the plasmid (data not shown), suggesting that the gene environment of bla NDM-1 in RP01 was likely identical to pNDM-HN380, a resistance plasmid distributed in Enterobacteriaceae isolates in China (Ho et al., 2012).

Discussion
Clinical isolates carrying bla NDM-1 have been associated with serious infection and high mortality, and the co-production of bla NDM-1 with other resistance genes always leads to the failure of antimicrobial treatment.Based on high throughput genome sequencing and ResFinder resistance gene analysis, the current report presents a description of a bla NDM-1 gene co-harboured with other 19 resistance genes in the rare pathogen R. planticola, highlighting the ability of bla NDM-1 to spread to unusual pathogens.