Type II and type IV topoisomerase mutations in clinical isolates of Morganella morganii harbouring the qnrD gene

Introduction The aim of this study was to show the emergence of the qnrD gene among fluoroquinolone-resistant Morganella morganii isolate. The occurrence of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC,parE) genes was also investigated in this strain. Methodology 95 clinical Enterobacteria were screened for harbouring the qnrD gene. The clinical isolate of M. morganii was recovered from urine from a patient hospitalized in the urology unit at Fattouma Bourguiba Hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method. Quinolone susceptibility was studied with microbroth dilution technique. The investigations of plasmid mediated quinolone resistance (PMQR) and topoisomerases mutations were performed by polymerase chain reaction and nucleotide sequencing. Results This isolate showed high level of resistance to quinolones. The MIC with microbroth dilution technique was 512 μg/ml for norfloxacin, 256 μg/ml for ofloxacin and ciprofloxacin and 64μg/ml for levofloxacin. This strain was found to harbour the quinolone resistance determinant qnrD. In addition, this strain harboured two new gyrB mutations (S463A, S464Y) and one parC mutation (S80I). Conclusions This is the first report in Tunisia of qnrD determinant and tow new gyrB muations in M. morganii. The nosocomial infection due to this proteeae invites further study of its epidemiologic evolution.


Introduction
The first qnr gene was observed in the late 1990s and described as a plasmid-mediated quinolone resistance gene (PMQR). Since that, five types of qnr genes have been reported: qnrA, qnrB, qnrC, qnrD, and qnrS. Their sequences are deposited at the following website (http://www.lahey. org/qnrStudies/). Most were observed in Enterobacteriaceae, located on large conjugative multi-resistant plasmids, such as pMG252 seen in the original qnrA1-positive strain [1].
We describe the new gyrB and parC mutations in a clinical isolate of M. morganii harbouring the rare qnrD gene that is first reported in Tunisia.

Case presentation
On 22 November 2012, a 76-years old male presented to the urology department with hematuria and burning micturition that were neglected by the patient since six months. On presentation, he was not febrile with normal vital signs and negative urine culture.
The ultrasonography shows a bladder of budding aspect, bifocal with long axis (3 cm). The histopathology revealed urothelial carcinoma infiltrating high-grade bladder (II pTa). On 30/11/2012, underwent a transurethral resection of bladder tumor. A double-current probe 18 c was also introduced.
On 19/01/2013 the patient presented an acute pyelonephritis and the urine culture showed the presence of quinolone resistant M. morganii. The empiric antibiotic therapy consisted of three doses a day of 2_g Ampicillin during 4 days. Urine culture subsequently yielded M.morganii. The antibiotic regime was then replaced with three doses a day of cefoxitin 1_g during one week. After this, the urine culture was negative but the haematuria still existed.

Bacterial strain
M. morganii was isolated from urine and identified using the Vitek 2 identification system (bioMérieux, Marcy l'Etoile, France). The antimicrobial susceptibilities of the M. morganii isolate were determined on Mueller-Hinton agar by the standard disk diffusion procedure as described by the Antibiogram Committee of the French Society for Microbiology (http://www.sfm.asso.fr). The microbroth dilution technique was used to determine the minimal inhibitory concentrations (MIC) for norfloxacin, ofloxacin, ciprofloxacin and levofloxacin.

Conjugation
The transferability of qnrD gene between the M. morganii isolate and the E. coli J53Az r recipient were carried out in LB broth. Cultures of donor and recipient cells in logarithmic phase (0.5 ml of each) were added to 4 ml of fresh LB broth and incubated overnight without shaking. Transconjugants were selected on Trypticase soy agar (TSA) plates containing sodium azide (100 μg/ml; Sigma Chemical Co., St. Louis, Mo.) for counterselection and gentamicin (10 μg/ml) to select for plasmid-encoded resistance.

Detection of mutations in the topoisomerase II and IV genes
The quinolone resistance-determinant regions (QRDRs) of gyrA, gyrB, ParC and parE was amplified as described previously [11]. Products resulting from amplifications were subjected to sequencing. Analysis and comparison of nucleotide and amino acid sequence data were carried out using Lasergene software (version 7.1; DNASTAR, Wisconsin, USA), and programs available from the national Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov).

Antibiotic susceptibility of M. morganii
The clinical strain of M. morganii showed a low level of resistance to beta-lactams due to its resistance to ampicillin, amoxicillin, ticarcillin and piperacillin only. It was susceptible to amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin/tazobactam, cafalotin, cefamandol, cefoxitin, cefotaxim, ceftazidim, cefpirom, aztreonam, imipenem and ertapenem. This strain was resistant to gentamicin and tobramycin but susceptible to amikacin. It showed also, decreased susceptibility to trimethoprime/sulfamides and rifampicin. The MIC with microbroth dilution technique was 512 μg/ml for norfloxacin, 256 μg/ml for ofloxacin and ciprofloxacin and 64 μg/ml for levofloxacin.

Detection of PMQR genes
The PMQR screening with PCR revealed the presence of qnrD in M.morganii only. None of the other studied Enterobacteria harboured the qnrD gene. The sequencing result showed the presence of qnrD1 in the M. morganii isolate. qnrA, qnrB, qnrS, qnrC, aac-Ib-cr and qepA genes were not found in this strain.

Conjugation
The conjugative reaction fails and the qnrD gene couldn't be transferred to E.coli J53.

Topoisomerase mutations
Sequencing of PCR products of QRDRs in gyrB and parC showed two mutations in the QRDR of gyr B at 463 and 464 codons. The first one is an S463A substitution and the second is an S464Y substitution.
Mutation was found also in the QRDR of parC at codon 80. It was an S80I substitution.

Discussion
The Gram-negative M. morganii is found in the environment and as a part of the normal flora of humans. It has been considered as a rare cause of human infections. However, it is an important opportunistic pathogen in urinary tract, soft tissue infections and also in infections following surgery [12,13]. In this case of study, the old age of the patient, the bladder tumor and its resection and the probe introduction after the surgery are risk factors leading to the urinary tract infection with the opportunistic M. morganii. This M. morganii strain was highly resistant to quinolones and qnrD was detected. qnrD was not found to be self transmissible and it could have spread either on a mobilizable plasmid or on a transferable structure integrated into a conjugative plasmid [6]. The transfer of quinolone resistance due to qnrD is always studied on transformants never on transconjugants. In the study of Hu et al., Proteus mirabilis harbouring qnrD failed to transfer quinolone resistance to E.coli 600 by conjugation [14]. The MIC of ciprofloxacin in E.coli DH10B cells increased in the transformants carring the plasmid pBR322 with the cloned qnrD from 0,002 μg/ml to 0,06 μg/ml (by a factor 32) [2].
However, qnrD positive clinical strains described so far exhibited a high level of quinolone resistance with ciprofloxacin MIC ranging from 1 to higher then 32 μg/ml and they are likely to harbour other quinolone resistance mechanisms [14,15]. Topoisomerase mutations have not been always investigated in qnrD positive clinical strains in order to evaluate how both mechanisms interact. The gyrA and parE primers used to amlify the QRDRs for Enterobacteria were not available for M. morganii. The study of Mazzariol A et al. couldn't to amplify gyrA and parC genes in this bacteria and this is due to its unknown genome [15].
To date, none of M. morganii was described with gyrB mutations. In this report, we described for the first time, two new gyrB mutations in M. morganii. In gramnegative bacilli, gyrB mutations are rare in clinical strains and they have been described at positions 426, 431, 447, 463, 464 and 466. The Ser 463Ala mutation has been linked to Klebsiella oxytoca [11]. This is the first report of the previous mutation in qnrD harbouring M. morganii. The Ser 464 Phe mutation has been linked to quinolone resistant S.enterica, P.aeruginosa and P.mirabilis [16][17][18]. In this study, the gyrB Tyr464 mutation was different from those described previously.
For M. morganii, the first ParC mutation was reported in the Tunisian study of Mahrouki et al. [19]. It was an S80I mutation associated to the qnrS 1 gene.
Another ParC mutation (S80R) was also described previously in this Proteeae [20]. In this study, we described the first S80I mutation in qnrD-positive M. morganii. This type of mutation was already observed in other Enterobacteria but never associated to the qnrD gene [21].
In summary, we have reported for the first time in Tunisia the rare qnrD in M. morganii and also found two new gyrB mutations (S463A and S464Y) and one parC substitution (S80I). In addition to qnrD gene, both mutations in gyrB and ParC could contribute to the high level of resistance to quinolones in this strain. The nosocomial infection caused by this bacterium invites further study of its epidemiologic evolution.

Competing interests
The authors declare that they have no competing interests.
Authors'contributions MNY carried the bacteria collection and typing antibiotic susceptibility, PCR screening, sequence blasting and writing the manuscript. IDR is project designer and co-supervisor of research work. QG Laboratory supervisor, she controlled the analysis, interpretation of data and controlled the manuscript. MM provided the necessary materials for collection, typing and doing the susceptibility tests. MA project designer and general supervisor of research group. MW General supervisor of research experiments and controlled the manuscript. All authors read and approved the final manuscript.