Characteristics of Klebsiella Pneumoniae ST4 Coharboring qnrB1, aac-Ib-cr, CTX-M-15 and SHV 11 in a Tunisian Hospital

Background: Multiresistant Klebsiella pneumoniae are predominant cause of hospital acquired infection. This work describes the molecular epidemiology of these isolates in Tunisian Hospital. Methods: Between October 2010 and June 2013, 50 non-duplicated clinical K.pneumoniae were selected on the basis of nalidixic acid (NA) resistance and were characterized. Isolates were identied using APi 20E system. Susceptibility testing was determined using the disc diffusion method and the microdilution technique to determine the MIC of ciprooxacin. PMQR and ESBL genes were detected by PCR and positive results were conrmed by direct sequencing of PCR products. Multilocus sequence typing (MLST) was performed to determine the genetic relationship among isolates. Conjugation and transformation were done to know if PMQR and ESBL were carried with one or two plasmids. Results: A total of 20 PMQR harboring K.pneumoniae representing 40% of all NA resistant isolates were characterized. Among PMQR positive K.pneumoniae 13 were resistant to amoxicilline, amoxicilline/clavulanic acid, ticarcilline, piperacilline, cefaloridine, cefotaxime (CTX) and ceftazidime. The rate of resistance to gentamicine, tobramicine and amikacine were 85%, 95% and 25% respectively. Out of 20 K.pneumoniae (60%) were qnr positive (1 qnrA6 and 11 qnrB1) and (60%) were aac-Ib-cr positive. 33.3% coharbored the aac-Ib-cr and qnrB1 determinants. Out of all PMQR positive strains, 65% harbored ctx-M-15 gene. It was associated to shv11 in three cases and tem1 in two cases. The predominant types were ST4 (35%) and ST15 (20%). ST 101(15%) and ST 147 (10%) come in second order. one case of each ST14,ST86,ST336 and ST307 were also observed. qnrB1, aac-Ib-cr,


Background
Pathogenic Klebsiella pneumoniae resistant to quinolones and β-lactam drugs is widely recognized as important bacteria causing array of diseases [1]. Antibiotic sensitivity assays have revealed high frequencies of quinolone drug resistance in extended spectrum beta lactamases (ESBL) producing clinical isolates [1]. The distribution of qnr and ESBL alleles varies among countries. In 1998, the rst plasmid-mediated mechanism of resistance to quinolones was described in multiresistant K.pneumoniae harbouring the qnrA gene on pMG252 plasmid and associated to the cephalosporinase Fox-5 [2,3]. Several studies have found the qnr determinants mainly qnrA and qnrB that were more frequent in ESBL producing Enterobacteriacae; the genes were carried by large plasmids  in Class1 integron type sul1,qnr S was more associated to tem-1 and lap-1 [4,5,6,7,8] The aac-Ib-cr gene was found in various integrons, especially on IncF11 plasmid expressing ctx-m-15 that have spread rapidly so that ctx-m-15 has become predominant ESBL in many countries around the world [9,10,11,12,13,14,15,16,17,18].
Very few studies have reported on the problem of the coexisting of PMQR and ESBL in K. pneumonie in Africa. [4,6] In Tunisia, qnrA6 and CTX-m-15 were the predominant allele among K.pneumoniae isolates [4,27,28]. qnrB1 coexisted with ctx-m-15, SHv-28 and Tem-1 in 125 Kb plasmid through the study of Dahmen et al in a Tunisian hospital [4].
Despite the high prevalence of these isolates in nosocomial infections, large studies to investigate the molecular epidemiology of these isolates in Africa are still lacking and the sequence type of coharboring PMQR and ESBL K.pneumoniae are not yet known.
We therefore conducted the rst epidemiological study in Africa to determine the molecular epidemiologie of PMQR and ESBL coproducing k.pneumoniae. This is also the rst report of aac-Ib-cr gene among K.pneumoniae despite its huge distribution in several countries.

Sample selection
In total, 50 non-duplicate clinical K.pneumoniae isolates recovered between October 2010 and June 2013 at Fattouba Bourguiba hospital (Tunisia) were selected on the basis of nalidixic acid resistance.

Antimicrobial susceptibility testing
The antibiotics susceptibilities of the K.pneumoniae isolates 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).
Positive results were con rmed by direct sequencing of PCR products.
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).

2-4 Multi locus sequence typing (MLST)
The genetic relationship among K. pneumoniae isolates was assessed based on the Multilocus Sequence Typing Method as described previously on the website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). PCR for seven housekeeping genes; rpoB, gapA, mdh, pgi, phoE, infB and tonB was conducted and products were directly sequenced. Analysis was carried out as described on the website.

Conjugation
The transferability of qnr, aac-Ib-cr and ctx-m-15 genes between the K.pneumoniae isolates 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.
To determine if quinolone and ESBL resistance were cotransferred, colonies were replicated onto TSA with and without cipro oxacin (0.06 _g/ml). MICs of cipro oxacine for the donor, recipient, and transconjugant strains were measured by E-test.

Transformation
For transformation reactions, Top 10 competent cells and plasmid extracted from transconjugants were taken to ice for 2 min. 5 to 10µl of plasmid were taken and added to 100µl of competent cells. The mixture was taken to ice for 30 min, then to 42°C for 1 min. After this it was taken to ice again for 3 min without mixing. After this step 900µl of LB broth were added to each mixture then taken to 37°C for one hour with cheeking.

3-1 Distribution of PMQR positive isolates
Out of 50 K pneumonia resistant to quinolones, 20 (40%) harbored PMQR over a period of three years. Table I characterize these isolates. During the survey period of quinolone resistance in K. pneumoniae, all of the strains had high MIC of cipro oxacine (16-1024µg/mL). A higher rate of resistance to commonly used non-quinolones was observed. Among PMQR harbouring K.pneumoniae isolates 13 were resistant to Amoxicillin, Amoxicillin/clavulanic Acid, ticarcillin, piperacillin, cefaloridine, cefotaxim and ceftazidim. As well as, they possessed an ESBL by phenotypic testing. The rate of resistance to gentamicin, tobramicin and amikacin were 85%, 95% and 25% respectively. Resistance to trimethoprime/sulfamides was 60%. All isolates were sensitive to imipenem using the disc diffusion method.
Among the twenty PMQR positive K.pneumoniae, 13 (65%) harbored the bla CTX-M-15 determinant. The previous gene was associated to the bla SHV11 gene in three cases and coexisted with the bla TEM1 gene in two cases.

3-3 Genetic relatedness
Seven ST4 K.pneumoniae variant were isolated in the Pediatric Unit. Four isolates belonging to ST15 were found in the nephrology unit. Three strains assigned to ST101 were recovered from the nephrology unit (n=2) and the ICU (n=1). Tow ST147 K.pneumoniae variant were also isolated from the ICU. One case of each ST14, ST86, ST336 and ST307 were isolated from Surgery, Padiatric, ICU and Cardiology respectively. All the conjugation reactions between Ecoli J53 and the donor 154 or the donor 2116 has given transconjugants able to give colonies on CTX+azide plates and Cip+azide plates.

3-4 PMQR and
However, conjugation reactions between E.coli J53 and the donor 5189 have given two types of transconjugants. First type: when inoculated from CTX+azide plate to Cip+azide plate they give colonies.
Second type gives colonies only on CTX+ azide plates.  contains two types of plasmids, the rst one is carrying the three cited genes (transformant E) and the second one is carrying aac-Ib-cr and CTX-m-15.

3-Discussion
The results of this study provide insights into the molecular epidemiology of PMQR and ESBL producing K. pneumoniae isolates in the university hospital Fattouma Bourguiba in Monastir, Tunisia, thereby representing the rst study to characterize PMQR and ESBL producing K. pneumoniae in Tunisia.
During our three years study, most of the K.pneumoniae was isolated from patients with urinary tract infection at inpatient department. Blood was the second site colonized by these bacteria. It was shown that multidrug resistant K.pneumoniae is one of the important nosocomial and community acquired opportunistic bacteria causing urinary tract infection, pneumonia, septicemia etc [1] The majority of the isolates characterized in this study were from pediatric units. Infections caused by PMQR and ESBL harboring organisms in neonates and pediatric wards are usually reported to be hospital acquired and associated with invasive procedures [38,39,40 ]. The predominance of these isolates in these units could be explained by the high use of the third generation of cephalosporins, as cefotaxim was the most prescribed drug in pediatric unit during the study period. Comparing to other units, the MIC of cipro oxacine (16-64µg/ml) was lower in pediatric wards as cipro oxacin is usually contraindicated in children and neonates. High rates of cipro oxacin resistance were detected in the ICU, the second ward contaminated with multidrug resistant K.pneumonia. These units have high rates of empirical treatment using uoroquinolones, 3 rd generation cephalosporins and also carbapenems.
Among quinolone resistant K.pneumoniae, 40% only harbored the PMQR genes. It has been demonstrated that quinolone resistance in Enterobacteriaceae family usually results from mutations in genes carried by chromosomally encoded type II topoisomerases, e ux pump or porin related protein [41].
PMQR such as qnr and aac(6')-Ib-cr may facilitate the spread and increase the prevalence of quinolone resistant strains. To date, qnr genes have been widely detected in Africa, Asia, America and Europe. The co-harbour of aac(6')-Ib-cr and qnr genes have been reported in several studies. In K. pneumoniae the aac(6')-Ib-cr gene was related to qnr B1 [6], to qnr B32 [5], to qnrS1, qnrB6 and qnrB4 [42].
It was also shown that PMQR and ESBL could be cotransferred in several studies and this is amplify the infection risk due to these strains of K.pneumoniae [1,4,5,42] This is the rst report in Tunisia of coexisting aac(6')-Ib-cr and qnrB1 in K.pneumoniae. Moreover, these two PMQR coexisted with bla CTX-m15 and shv11 in three cases and with ctx-m15 and tem1 in tow cases. To the best of our knowledge, this is the rst report of coexisting qnrB1, aac-Ib-cr, ctx-m-15 and shv11/tem1 in K.pneumoniae. Referring to conjugative reactions, qnrB1, aac-Ib-cr and ctx-m15 were co transferred from tow donors.
Plasmids extracted from the corresponding transformants were the same in size and there were only one plasmid for each transformant. This is con rming that these three genes were carried by the same plasmid. The third donor gave transconjugants with qnrB1, aan-IB-cr and ctx-m-15 or transconjugants with aac-Ib-cr and ctx-m-15 only. This is had already in uence on MIC of cipro oxacine on transconjugants. Effectively, the MIC of cipro oxacine on transconjugants with qnrB1 and aac-Ib-cr genes was higher (0.5µg/ml) then those with aac-Ib-cr only (0.064µg/ml). This is mean the expression of qnrB1 in resistance to cipro oxacine is match more important than the expression of aac-Ib-cr. The transformation of top 10 with plasmid exracted from transconjugants coming from the third donor has shown tow types of transformants; the st type is carrying one plasmid harboring the three cited genes (transformant E), the second type is carrying one smaller plasmid harboring aac-Ib-cr and ctx-m-15. Referring to the previous data the donor 1 and 3 are harboring one plasmid that carry 3 genes and the third donor harbor tow type of plasmids the rst carry the 3 genes and the second carry aac-Ib-cr and ctxm-15. Bought type of donor with one or tow plasmid are prohibiting the patient health and this is need important survey of the epidemiology of these strains. This is the rst epidemiological report in Tunisia using MLST to type strains of K.pneumoniae harboring PMQR. The results revealed mainly sequence type 4, 15,101 and 147 compared to previous reported ST 101, ST 107 and ST147 [27]. There is a signi cant distribution of the ST and the corresponding unit. The ST4 K.pneumoniae were found in pediatric wards, the ST 15 in nephrology unit, ST147 in ICU and the ST101 between the nephrology and the ICU. This phenomenon can be explained by the nosocomial spread of strains with the same sequence type among patients of the same unit. The study of Filippa et al, have demonstrated the spread of an unusual K.pneumoniae isolated from Nigerian patient among 10 French patients in the ICU of French hospital [6]. The ST4 and ST 15 K.pneumoniae were rst described in France, but until 2013, the ctx-m-15 doesn't exist in France and it was introduced among this country because of a strain coming from Nigerian patient [6]. The nosocomial contamination between patients could explain the harbor of new genes in ST4 and ST15 K.pneumoniae. The ST101 and ST147 K.pneumoniae were rst reported respectively in Poland and Hungary. Since that these sequence type have been reported to occur worldwide and are associated with multidrug resistant K.pneumoniae [43,44]. The ST14 found in the surgery unit was described previously in Africa [28]. The ST86, ST336 and ST307 are strange to Africa and this is the rst report of these types in Tunisia since they were described respectively in France, America and Netherlands. Tunisia has important relationship with the cited countries and this can explain the rst appearance of these ST types K.pneumoniae.

Conclusion
This study highlights the need to improve the antimicrobial resistance surveillance in Tunisia for K.pneumoniae to monitor the new types of resistance mechanisms emerging in different ward in the same or different hospitals. As well as, the factors responsible for the selection and dissemination of the high conjugative plasmids encoding the qnr genes, aac-Ib-cr determinant and different ESBL must be considered for the antibiotic policies and controlled to prevent other outbreaks in future.   Plasmid pro le extracted from transformants.

Supplementary Files
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