Detection of High Prevalence of TEM / SHV / CTX-M Genes in ESBL Producing and Multidrug Resistant Klebsiella pneumoniae and Klebsiella oxytoca

Background: Klebsiella spp. are Enterobacteriaceae frequently isolated from pathological specimens during urinary tract infections, bloodstream infection, and pus. They are becoming more and more resistant to antibiotics and challenging treatment options. β-lactamases are a great variety of enzymes capable of inducing resistance to βlactams. The objective of this study was to identify extended-spectrum-β-lactamase (ESBL) genes in Klebsiella spp. strains isolated from various specimens in Lomé, Togo. Methods: Sixty-four strains of Klebsiella spp. were isolated from different pathological specimens. They were then further characterized and tested against 3rd generation cephalosporin (ceftazidime, ceftriaxone, cefotaxime) and aztreonam. The detection of blaTEM, blaSHV and blaCTX-M was performed on these strains using simplex and multiplex PCR techniques. Results: Fifty five (85.94%) Klebsiella pneumoniae and 9 (14.06%) Klebsiella oxytoca were isolated. These strains derived from urine (n=33; 51.56%), vaginal swabs (n=21; 32.81%), pus (n=8; 12.5%) and sperm samples (n=2; 3.13%). All strains were resistant to cefepime. The resistance rate to other β-lactams was 29.69% (19/64) for piperacillin-tazobactam, 23.08% (12/52) for cefoxitin and 1.56% (1/64) for imipenem. Other inactive antibiotics were trimethoprim-sulfamethoxazole 96.72% (59/61), doxycycline 92.06% (58/63), ciprofloxacin 90.63% (58/64), nalidixic acid 80.95% (51/63), chloramphenicol 77.42% (48/62) and gentamicin 76.69% (51/64). Amikacin and fosfomycin remained the most active antibiotics with 1.56% (1/64) and 4.69% (3/64) resistance rates respectively. ESBL genes were detected in 63/64 (98.44%) strains. TEM/SHV/CTX-M was predominant 61.90% (39/63) followed by TEM/SHV 20.63% (13/63), SHV/CTX-M 11.11% (7/63), TEM/SHV 4.76% (3/63) and TEM 1.59% (1/63). Conclusion: ESBL genes occur more by combination 96.88% (62/64) than singularly 1.59% (1/63). These strains were also very resistant to quinolones and trimethoprim-sulfamethoxazole. These findings are of high importance in a medical and scientific perspective and may motivate decision makers towards a better monitoring and control of antimicrobial resistance in Togo.


Introduction
β-lactamases are serine enzymes (class A, C or D of Ambler classification) or metalloenzyme (Class B of Ambler classification) that confer resistance to β-lactams by hydrolyzing their β-lactam ring [1].The discovery and use of new classes of β-lactams have immediately been followed by the emergence of new β-lactamases.The first reported β-lactamases TEM-1/2 and SHV-1, described since 1960 from E. coli and Klebsiella pneumoniae were usually chromosomal.They are able to inactivate penicillins (amoxicillin, ampicillin, ticarcillin) hence the name penicillinases narrow spectrum [2].From the 80s, plasmid mediated extended-spectrum-β-lactamases (ESBLs) have been described and confer resistance to penicillins, oxyimino cephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cefepime) and monobactams (aztreonam) [3][4][5].These ESBLs are divided into several groups; the main ones are the derivated of TEM and SHV groups and CTX-M [6].In recent years, the CTX-M ESBL spread around the world and have been described in the commensal flora and also in hospital-acquired and community infections with a tendency to supplant the first ESBL groups TEM and SHV [7][8][9][10][11][12][13][14].
the most medically important species, often isolated from severe infections in hospitalized or ambulatory patients [25][26][27][28][29]. Klebsiella spp.are naturally resistant to penicillins (amoxicillin, ampicillin, piperacillin and ticarcillin) due to a low production of a class A chromosomal β-lactamase (penicillinase).The severity of Klebsiella spp.infections is increased when the clinical strains acquire resistance genes, such as ESBLs genes, following the misuse of antibiotics and other risk factors such as a long hospitalization associated with regular antibiotics, poor adherence, and low socioeconomic conditions [30][31][32].Previous studies have shown high frequency of ESBL in hospitals among Klebsiella spp. with variation from one continent to another.The global multi-center surveillance study Tigecycline Evaluation and Surveillance Trial (TEST) reported from 2004 to 2009 an overall prevalence of ESBL producing Klebsiella pneumoniae isolated from intensive care units (ICUs), ranging from 12.8% in North America to 26.6% in Europe, 33.8% in the Middle East, 35.6% in the Asia-Pacific region, 45.5% in Latin America an up to 54.9% in Africa [33].In addition, Hoban et al. reported a prevalence of ESBL of 8.8% and 38.9% in North America and Europe respectively with a predominance of CTX-M15 subgroup [34] on Klebsiella pneumoniae strains isolated from urinary tract infections.
In Togo, the prevalence of ESBL was previously reported [35]; however, their molecular characterization was not yet described.A recent study focused on the molecular characterization of ESBL producing E. coli (Salah et al. submitted for publication).The present study aimed to characterize ESBL genes in Klebsiella spp.isolated from various pathological specimens received at the National Institute of Hygiene in Lomé, Togo.

Collection and identification of Klebsiella spp. strains, antimicrobial susceptibility testing, and detection of ESBL phenotype
Klebsiella spp.strains were collected during a prospective study from May 2013 to July 2015 in the bacteriology laboratory of the National Institute of Hygiene (INH) in Lomé, Togo.This institute is a public health service specialized in biomedical analysis, epidemiological surveillance, immunization, water, and food quality control.The strains were isolated from various pathological specimens including urine, vaginal swabs, pus, and sperm samples.The standard microbiological methods were used to isolate and purify bacterial strains on Mac-Conkey or eosin methylene blue (EMB) media.The strains were identified by the API 20E identification system (Biomérieux, Marcy-Etoile, France).

Extraction of bacterial DNA
The bacterial chromosomal and plasmid DNA was extracted by a boiling method.Briefly, from the TSB, strains were reactivated on tryptic soy agar (TSA) for 18-24 h and then inoculated in Luria Bertani broth (LB, 2 ml).After 18-24 h culture, LB broth were centrifuged (10 000 RPM/min for 10 min) and bacterial cells were suspended in 500 μl of phosphate buffer (100 mM, pH 7) to weaken the membranes.An immersion in a boiling water bath (100°C for 15 min) releases the genetic material.The DNA is then precipitated with 250 μl of absolute alcohol, washed twice in 1000 μl of 70% alcohol (stored at -20°C), dried and re-suspended in 100 μl of sterile water.
An Electrophoretic migration at 100 volts for 1 hour was performed on a 2% agarose gel stained with ethidium bromide to separate PCR products.A marker of 100 bp was used as reference.After migration, the various bands were observed under UV illumination and pictures recorded.

Statistical analysis
Statistical analysis were performed with the software Epi Info Version 7.1.1.14.Fisher's exact test with a significance level of 5% (p<0.05) which was used to interpret results.

Ethical consideration
This study received the INH approval for the transfer of Klebsiella spp.strains, to the molecular biology laboratory of CERBA/ LABIOGENE, University Ouaga I, Professor Joseph Ki-Zerbo, Burkina Faso.The institutional ethic committee of CERBA/LABIOGENE reviewed and approved the study protocol.
All Klebsiella pneumoniae strains were resistant to ceftazidime, cefepime and aztreonam.The resistance to ceftriaxone and cefotaxime was 98.18% (54/55).All (100%) Klebsiella oxytoca strains were resistant to ceftriaxone and cefepime and 88.89% (8/9) were resistant to ceftazidime, cefotaxime and aztreonam.The resistance profile to other β-lactams and other antibiotics is presented in Figure 1.Only imipenem, amikacin and fosfomycin are very active on Klebsiella spp.strains with a low resistance rate (below 5%).Ciprofloxacin, doxycycline and trimethoprim-sulfamethoxazole are the most inactive antibiotics with at least a resistance rate of 90%.

ESBL genes distribution within Klebsiella spp. strains
With regards to the PCR results, bla TEM , bla SHV and bla CTX-M group 1 (including bla CTX-M 1, bla CTX-M 3 and bla CTX-M 15) were found in 98.44% (63/64) Klebsiella spp.strains.Of these, 98.41% (62/63) strains harbored a combination of bla TEM , bla SHV and bla CTX-M with predominance for the triple combination of TEM/SHV/CTX-M estimated at 61.90% (39/63) (Figure 2).Only one strain of Klebsiella oxytoca carried TEM gene.This strain was negative to the double disk test synergy for the phenotypically detection of ESBL.We did not identify any gene in one Klebsiella pneumoniae strain that did not show any ESBL phenotype.

Distribution of the combination TEM/SHV/CTX-M according to the biological specimen
The combination of TEM, SHV and CTX-M genes was carried by 57.14% (12/21), 45.45% (15/33) and 37.5% (3/8) of Klebsiella spp.strains isolated from vaginal swabs, urine and pus respectively.Klebsiella pneumoniae from the two sperm samples expressed simultaneously these three genes, TEM, SHV and CTX-M.

Discussion
Nowadays, resistance to antibiotics is a real public health concern worldwide.It has been clearly demonstrated that the production of βlactamases is the most important mechanism of antibiotic resistance in Gram negative bacteria.The description of first β-lactamases TEM-1/2, SHV-1 and their derivatives was followed by the characterization of new ones dominated by CTX-M group which is now, the most widespread.This study was carried out to characterize ESBL genes in Klebsiella spp.strains resistant to at least one 3rd generation cephalosporins or aztreonam, isolated from different specimen received at INH Lomé, Togo.It has been reported that Klebsiella spp.present a high medical interest among other Enterobacteriaceae as expressing ESBL resistance mechanism and are frequently isolated in hospital and community infections.
According to our expectation, all Klebsiella spp.strains were highly resistant (over 98%) to 3rd generation cephalosporins (ceftazidime, ceftriaxone, cefotaxime), 4th generation (cefepime) and aztreonam.However, their resistance rate is higher than those found in previous studies conducted in North America.Indeed, through the international monitoring program for antibiotic resistance SENTRY involving four geographic regions, Asia Pacific, Europe, Latin America, North  [41].For piperacillin-tazobactam and cefoxitin two β-lactams subject of comparative study on their efficiency on ESBL producing Enterobacteriaceae [42][43][44], we found a prevalence of resistance at 30.91% and 20.93% for Klebsiella pneumoniae and 22.22%, 33.33% for Klebsiella oxytoca respectively.These prevalence rates seem low compared to those reported in North America, 47.4% for piperacillintazobactam, and 44.2% for cefoxitin [40].The resistance to cefoxitin can be induced by a production of cephalosporinase AmpC, a class A enzyme that inactivates other 3rd generation cephalosporins.AmpC decreases the intracellular concentration of the antibiotic by the efflux pump mechanism and mediates a loss of outer membrane porins [45].
Fortunately, Klebsiella spp.isolated in this study have remained relatively sensitive to imipenem, amikacin and fosfomycin which are among other, recommended antibiotics in case of infection by ESBL producing bacteria [46][47][48].The prevalence of resistance to these antibiotics ranged from 0 to 5% as in a series of ESBL producing Klebsiella spp.strains isolated in Tunisia, where the prevalence of resistance to imipenem was zero [49].However, the resistance rates for amikacin and fosfomycin were high, 10% and 17.5%, respectively [49].Higher resistance rates were found in non-intensive care units in the United States of America and Europe for imipenem (22% and 6.9%) and amikacin (18.9% and 19.1%), respectively [50].
In addition, Klebsiella spp.isolated in the present study showed a strong resistance rate to quinolones (nalidixic acid and ciprofloxacin) ranging from 78.18% (43/55), 89.09% (49/55) for Klebsiella pneumoniae to 100% for Klebsiella oxytoca.Similar results were found in Congo, in Tunisia and in Bolivia.In Congo, 83.3% of Klebsiella spp.
The analysis of the PCR product showed the simultaneous presence of the three genes, TEM, SHV and CTX-M in 61.90% of the studied strains, 59.26% (32/54) in Klebsiella pneumoniae, 77.78% (7/9) in Klebsiella oxytoca.TEM was detected in 1.59% (1/9) Klebsiella oxytoca strain, SHV gene alone was not found in this study.In Burkina Faso, on 28 ESBL producing Klebsiella pneumoniae screened for the detection of bla genes, 14.28% expressed TEM, 10.71% SHV, 25% CTX-M, 10.71% TEM and SHV and 3.57% of strains exhibited the triple combination of TEM, SHV and CTX-M [52].In another study conducted in Burkina Faso, CTX-M15 was found in 17/17 Klebsiella spp.isolated from urine in a pediatric hospital in Ouagadougou [53] suggesting that CTX-M15 was present in West Africa.Our results are consistent with those found by Alibi et al. in Tunisia, on a large series of 118 ESBL producing Klebsiella spp.where the triple combination TEM/SHV/CTX-M represented 44.91% (53/118), and the double combinations were SHV/CTX-M represented 28.81% (34/118) of cases, TEM/CTX-M represented 4.23% (5/118) and TEM/SHV represented 3.39% (4/118) of cases [54].The presence of TEM, SHV and CTX-M genes alone in the study of Alibi et al. in [57].It is observed that the expression of bla CTX-M is currently more prevalent and as we report for the first time in Togo, more frequent in a triple combination with bla TEM and bla SHV .The explanation of this triple combination should be more investigated.
The antimicrobial susceptibility profile of ESBL producing Klebsiella spp.strains shows that the different ESBL genes do not significantly influence the activity of the antibiotics.As expected, resistance to imipenem, amikacin and fosfomycin was low.On the contrary, the resistance rates to trimethoprim-sulfamethoxazole, ciprofloxacin, nalidixic acid, chloramphenicol and gentamicin were very high ranging from 65.63% for chloramphenicol in the triple combination TEM/SHV/CTX-M to 100% for most of them in all double combinations.These antimicrobial resistances associated with ESBL genes are in line with our expectation and are consistent with the results found in previous studies [1][2][3][4][5][6].Indeed, it has been described that ESBL genes and some antibiotic resistance genes are often found on the same mobile genetic elements and are thereby transmitted together by horizontal transfer [1][2][3][4][5][6].In Tanzania, on ESBL producing Klebsiella pneumoniae causing sepsis in neonates, Mshana et al.
showed that bla CTX-M 15 was localized on a plasmid of 25 to 485KB and that ST14 and ST48 clones carried also resistance genes to gentamicin and trimethoprim-sulfamethoxazole [56].More recently, in a study in Central African Republic, Rafai et al. showed that all ESBL producing Klebsiella pneumoniae isolated from surgical wounds, expressed bla CTX-M 15 and aac(6 H)-Ib-cr [58].The last one has been described to be associated with a resistance to ciprofloxacin, norfloxacin and aminoglycosides [59].This is probably due to the same mechanisms that might explain the results we observed.
In this study, the combination of TEM, SHV and CTX-M genes was found on strains isolated mostly from urine and sperm.This result is also consistent with previous findings in Tunisia where 86.44% of ESBL producing Klebsiella spp.were isolated from urine and 44.91% (53/118) of all studied strains expressed the triple combination TEM/SHV/CTX-M [54].The observation of this combination based on the pathological sample needs to be analyzed more thoroughly.

Conclusion
In this study, we observed that the majority of Klebsiella spp.resistant to 3rd generation cephalosporins were producing ESBL.The major groups of ESBL genes, TEM, SHV and CTX-M were present.We report for the first time, that the triple combination of TEM, SHV and CTX-M genes was predominant.Stressfully, all ESBL producing Klebsiella oxytoca were resistant to quinolones (nalidixic acid and ciprofloxacin), doxycycline and trimethoprim-sulfamethoxazole and also to a lesser extent to gentamicin and chloramphenicol.The same trend is observed for ESBL producing Klebsiella pneumoniae.
Antibiotics that remained active, including fosfomycin, amikacin and imipenem are unfortunately a little affordable to patients in limited resources countries.It is therefore important to these countries to strengthen public health policies in order to prevent, monitor and control antimicrobial resistance.In addition, other studies have been done for screening of medicinal plants with potential antimicrobial activities.In a public health point of view, it is now important for political, economic and medical sectors to work together towards the selection of those plants which could be active against clinical resistant strains.

Figure 2 :
Figure 2: Distribution of bla genes among Klebsiella spp.strains

Table 1 :
Sequences of targeted bla genes.
Diagbouga S, Salah FD, Sadji AY, Dabire AM, Nadembega C, et al. (2016) Detection of High Prevalence of TEM/SHV/CTX-M Genes in ESBL Producing and Multidrug Resistant Klebsiella pneumoniae and Klebsiella oxytoca.J Clin Diagn Res 4: 129.combination TEM/SHV/CTX-M to 66.67% for the double combination TEM/SHV in Klebsiella pneumoniae.Similarly, the resistance rate to cefoxitin ranged from 17.39% for the triple combination TEM/SHV/CTX-M to 33.33% for the double combination TEM/SHV.We did not find statistically significant differences for these two antibiotics (p=0.472 and p=0.426>0.05) between the groups expressing three ESBL genes (TEM/SHV/CTX-M) or two ESBL genes (TEM/SHV, SHV/CTX-M, TEM/CTX-M). Citation: