Abstract
Purpose: The study aimed at assessing any association between quinolone resistance, MDR and ESBL production and their relation with the presence of integrons in Esherichia coli and Klebsiella pneumoniae.
Methods: E.coli and K.pneumoniae isolated from various clinical infections were fully identified and analyzed for being quinolone resistant. These isolates were further tested for ESBL production, multi drug resistance and carriage of integrons.
Results: In total, 135 isolates were confirmed as quinolone resistant. K.pneumoniae was observed as potent ESBL producer in comparison to E.coli. Ciprofloxacin resistance in both organisms was related significantly with the presence of integron class 1, co-presence of class 1 and 2 as well as to the presence of ESBL production (p< 0.001). However, nalidixic acid resistance was related significantly (p< 0.01) with only integron class 1 and to the presence of ESBL production. Class 1 and 2 integrons were found in 73.5% of MDR isolates with 13.2% of them possessing both intI1 and intI2 genes.
Conclusion: Prevalence of quinolone resistance together with ESBL production and MDR in E.coli and K.pneumoniae has contributed to the emergence of antibacterial resistance burden. The higher integron prevalence in our isolates advocates the potentiality of these isolates as a source for dissemination of resistance determinants.
Keywords: Quinolones, Escherichia coli, Klebsiella pneumoniae, ESBL, Multi-drug resistance, Integron
Introduction
In contradiction of nalidixic acid, which is used only for urinary infections, the fluoroquinolones (FQ) have a broad range of therapeutic indications and in fact, were a major therapeutic advance of the 1980s.1 Nevertheless, recent years have witnessed FQ resistance in Escherichia coli (E.coli) and other Enterobacteriaceae,2 contingent on multiple mutations that diminish the affinity of its topoisomerase II and IV targets in various ways.3 Alarmingly, however, is the upward trend observed in last two decades in the co- occurrence of ciprofloxacin resistance with resistance to beta-lactam antibiotics in E.coli and Klebsiella pneumoniae.4-7 Furthermore, emergence of multi drug resistance (MDR) and Extended-Spectrum β-Lactamase (ESBL) production in E.coli and Klebsiella pneumoniae8 in chorus with FQ resistance has knockdown the infrastructure of therapy substantially.9 As regards to development of antibiotic resistance, the dissemination of resistance genes among bacterial strains is being debated frequently. One type of dynamic force which is perceived as a major crisis is located on the bacterial chromosome or a plasmid, and named as an integron helps bacteria to acquire novel combinations of resistance genes10,11 and disseminate them along with the emergence of MDR strains.10,12-14 Five integron classes related to antibiotic resistance have been described based on the homology of their integrase genes and Class 1 integrons are the most commonly found in nosocomial and community environments, followed by class 2 ones.15 The prevalence of integrons is high among gram-negative isolates from patients in Europe.14 Similar reports are available from Asian and Middle East countries.16-18
Our hospital is a tertiary University teaching institution, and a reference health care center for North West Iran. Similar to any hospital elsewhere worldwide, E. coli and K. pneumoniae are the two most isolated organisms from patient’s samples in the hospital’s microbiology section and for the last five years an upward trend in the incidence of quinolone resistance was observed in these clinical isolates. Thus, this study was taken up to uncover the level of association between quinolone resistance, MDR and ESBL production with the presence of integrons, in E.coli and K.pneumoniae isolated from a selected high risk groups of hospital admitted patients and those being referred as outpatients. To the best of our knowledge, this is the first report on the association between presence of integrons in quinolone resistant, ESBL production and MDR E.coli and K.pneumoniae from North West Iran.
Materials and Methods
Bacterial isolates and Antibiotic susceptibility testing
During a period of nine months, 234 E.coli and K.pneumoniae isolated from various clinical infections were fully identified according to standard bacteriological procedures.19,20 Duplicate isolates from the same patient were excluded. These isolates were subjected to routine antibiotic susceptibility testing performed by disc agar diffusion method.21 The antibiotics included were gentamicin (10μg), amikacin (30μg), ceftriaxone (30μg), ceftazidime (30μg), imipenem (10μg), co-trimoxazole (1.25μg), nalidixic acid (30μg), ciprofloxacin (5μg), cefamandole (30μg) and ceftizoxime (30μg) (Mast Diagnostics, UK). MDR was defined as resistance to 3 or more different group of antibiotics. FQ and nalidixic acid resistance was confirmed for non-susceptibility by minimum inhibitory concentration (MIC) on E-test (Liofilchem) performed according to manufacturer’s instructions with interpretative criteria of Clinical Laboratory Standards Institute (CLSI).21 Any decrease in the zone sizes for the 3rd generation cephalosporins was used presumptively as ESBLs producer, and was confirmed later by CLSI criteria.21 ATCC 25922 E. coli reference isolate served as the standard drug-susceptible control for disk diffusion and MIC measurements. The strains were preserved at －70°C in nutrient broth containing 15% v/v glycerol.22
Phenotypic ESBL confirmatory method
Antibiotic disks of ceftazidime (30 μg) with ceftazidime/clavulanic (30/10 μg), cefotaxime (30 μg) with cefotaxime /clavulanic acid (30/10 μg), cefpodoxime with cefpodoxime /clavulanic acid and aztreonam (30μg) (Mast Diagnostics, UK) were placed onto pre-inoculated Muller-Hinton agar plate with the test organism according to CLSI.21 Regardless of the zone diameters, a >5 mm increase in a zone diameter for an antimicrobial agent tested in combination with clavulanic acid versus its zone size when tested alone, indicated a probable ESBL production.21 ESBL producing strain K. pneumoniae ATCC 700603 and non-ESBL producing strain E. coli ATCC 25922 were used as positive and negative control in each test, respectively.
DNA extraction and integrase analysis
For DNA extraction, E. coli and Kpneumoniae were cultured in Lauria Bertani (LB) broth at 37°C overnight, and DNA was extracted by CTAB method.23 For detection of integrons, amplification of the integrase genes of class 1, 2 and 3 integrons (intI1 , intI2 and intI3) with the Int1F ⁄ Int1R ,Int2F ⁄ Int2R and Int3F/ Int3R primers as multiplex PCR was performed as described earlier to yield a PCR product of 475bp, 789bp and 922 bp respectively.24
Data were analyzed using the statistical package for social sciences (SPSS 18.0, IBM SPSS, New York, USA). Contingency table analysis was done by a chi-square test or two-tailed Fisher’s exact test where applicable. A p-value of less than 0.05 was considered as statistically significant. Pearson’s correlation was used to calculate association between antibiotics for detection of ESBL.
Results
Bacterial isolates
Two hundred and thirty four isolates obtained from outpatients (n=88) and inpatients (n=146) including, 150 (64.1%) E.coli and 84 (35.89%) K.pneumoniae were taken into study. E.coli was the predominant organism in the urine specimen and isolated more frequently in outpatients than K.pneumoniae, the two-tailed P value equals 0.0268 and the association was not found statistically significant. On the other hand, K.pneumoniae was the most frequently isolated bacteria in blood cultures from inpatients, though association not considered to be significant. No significant difference was observed in the prevalence of either or both pathogens from other clinical specimens (Table 1).
Table 1. Type of clinical specimens and distribution of E.coli and K.pneumoniae .
| Clinical specimens | Inpatients | Outpatients | ||
| E.coli | K.pneumoniae | E.coli | K.pneumoniae | |
| Blood | 14 | 27 | 8 | 7 |
| Urine | 53 | 22 | 57 | 9 |
| Wound | 10 | 11 | 4 | 3 |
| Endotracheal tube | 1 | 3 | 0 | 0 |
| Broncho- alveolar lavage | 1 | 2 | 0 | 0 |
| Catheter tip | 1 | 0 | 0 | 0 |
| CSF | 0 | 0 | 0 | 0 |
| Sputum | 0 | 0 | 0 | 0 |
| Body fluids | 1 | 0 | 0 | 0 |
| Total | 81 | 65 | 69 | 19 |
Quinolone resistance and MDR
On disk diffusion assay, E.coli and K.pneumoniae isolates obtained from urine specimens were found resistant (n=125; 88.65%) or intermediately resistant (n=16; 11.34%) to nalidixic acid, while 134 (71.05%) isolates, including 63 (47.01%) K.pneumoniae and 71 (52.98%) E.coli, irrespective of clinical source, were observed resistant to ciprofloxacin by disk agar diffusion method. In order to quantify this quinolone resistance, the MIC of ciprofloxacin and nalidixic acid was determined by E-test. MICs of nalidixic acid ranged from 8 to >256mg/L and for ciprofloxacin from 0.032 to >32mg/L. MIC50 and MIC90 values of nalidixic acid for 125 E.coli and K.pneumoniae isolates were found in resistance breakpoints (both = 163.55mg/L). Intermediate resistant isolates on disk agar diffusion were further confirmed as susceptible with MIC being <16mg/L. Ciprofloxacin MIC50 and MIC90 was observed as 24.78mg/L. One E.coli isolate found intermediate resistant on disk agar diffusion assay was later confirmed resistant by E-test, thus in total 135 isolates were found as quinolone resistant. The clinical source of these 135 quinolone resistant isolates were urine [75 (55.55%) E.coli, 15 (11.11%) K.pneumoniae], blood [7 (5.85%) E.coli, 18 (13.33%) K.pneumoniae], and wound [6 (4.44%) E.coli, 7 (5.18%) K.pneumoniae]. One E.coli obtained from bronchial secretion was also FQ resistant. Among these quinolone resistant isolates, 56 E.coli, and 57 K.pneumoniae were recovered from inpatients and 16 E.coli and 6 K.pneumoniae from outpatients.
Of 72 quinolone resistant E.coli isolates, 57 (79.16%) were highly resistant to 3rd generation cephalosporins, non-susceptibility being in the range of 49.2% - 85.9% [cefamandole (85.9%), ceftriaxone (81.6%), ceftazidime (63.3%) and ceftizoxime (49.2%)] followed by gentamicin (61.9%) and co-trimoxazole (61.6%), while this trend was more heavily observed in K.pneumoniae, with cephalosporin non-susceptibility appearing in the range of 52.3%- 96.8% [cefamandole (96.8%), ceftriaxone and ceftazidime (88.8%), ceftizoxime (52.3%)] followed by gentamicin (63.9%) and co-trimoxazole (60.3%). Cefepime resistance was low in E.coli (28%) as well as in K.pneumoniae (23%). Forty seven of 63 (74.60%) K.pneumoniae and 59 of 72 (81.94%) E.coli were simultaneously resistant to other antibiotics appearing as multi drug resistant isolates (n=106; 78.51%). The most frequent phenotype pattern of MDR was ceftazidime-ceftriaxone-cefamandole-ceftizoxime-gentamicin- cotrimaxazole found in 56 (52.83%) isolates, followed by ceftazidime-ceftriaxone-cefamandole- ceftizoxime- amikacin- gentamicin-cotrimaxazole in 31 (29.24%) and ceftazidime-cefamandole-ceftriaxone in 12 (11.3%) isolates. Phenotypic resistance pattern cefamanadole-ceftriaxone-ceftizoxime-amikacin-gentamicin-imipenem phenotype was disclosed by 7 (6.6%) isolates.
Quinolone resistance and ESBL
When quinolone resistant E.coli and K.pneumoniae isolates were tested by disk agar diffusion method for being ESBL producers, K.pneumoniae was observed as potent ESBL producer (n=44/63; 69.84%) in comparison to E.coli (n=42/72; 58.33%). However, the two-tailed P value equaled 0.2096 and the association was not found statistically significant. Among the beta lactams used alone and in combination for detection of ESBL production, ceftazidime, cefotaxime, cefpodoxime and cefotaxime combinations with clavulanic acid correlated well for detection of ESBL production in both bacteria and this correlation analyzed by Pearson’s correlation was found significant at the 0.01 level. However, ceftazidime was observed the most suitable substitute over cefpodoxime and cefotaxime alone and in combination with clavulanic acid (p<0.05) for detection of ESBL production. E.coli were highly resistant towards cefpodoxime (92%), aztreonam (78%), cefotaxime (69%), ceftazidime (66.1%) while, K.pneumoniae in comparison to E.coli revealed high resistance against cefpodoxime and aztreonam (90%) , ceftazidime (87%) and cefotaxime (85%). Table 2 shows the antimicrobial susceptibility of quinolone resistant E.coli and K.pneumoniae.
Table 2. Association of resistance to various antimicrobial agents and presence of integrase genes in E.coli and K. pneumoniae isolates .
| Antibiotics | Quinolone resistant E.coli (n=72) | Quinolone resistant K.pneumoniae (n=63) | ||||||||
| intI1 (%) | intI2 (%) | intI1,2 (%) | ESBL (+) a | ESBL (-)b | intI1 (%) | intI2 (%) | intI1,2 (%) | ESBL(+)a | ESBL(-)b | |
| Ciprofloxacin | 15 (26.7) |
11 (19.6) |
10 (17.8) |
36 (64.2) |
20 (35.7) |
23 (37.8) |
10 (16.3) |
4 (6.5) |
42 (68.9) |
19 (31.1) |
| Nalidixic acid | 17 (34) |
7 (14) |
10 (20) |
30 (60) |
20 (40) |
8 (50) |
3 (18.7) |
1 (6.2) |
12 (75) |
4 (25) |
| Amikacin | 3 (21.4) |
3 (21.4) |
6 (42.8) |
14 (100) |
---- | 4 (33.3) |
3 (25) |
3 (25) |
9 (75) |
3 (25) |
| Gentamicin | 16 (34) |
9 (19.1 |
9 (19.1) |
34 (72.3) |
13 (27.6) |
19 (47.5 |
10 (25) |
4 (10) |
34 (85) |
6 (15) |
| Cotrimoxazole | 16 (32.6) |
8 (16.3) | 9 (18.3) |
29 (59.1 |
20 (40.8) |
21 (53.8) | 10 (25.6) |
5 (12.8 |
32 (82) |
7 (17.9) |
| Cefamandole | 21 (30.4) |
11 (15.9) |
10 (14.4) |
42 (60.8) |
27 (39.1) |
24 (38) |
11 (17.4) |
4 (6.3) |
44 (69.8) |
19 (30.1) |
| Ceftizoxime | 11 (30.5) |
4 (11.1) |
6 (16.6) |
22 (61.1 |
14 (38.9 |
13 (37.1 |
7 (20) |
4 (11.4 |
28 (80) |
7 (20) |
| Ceftazidime | 21 (40.3) |
9 (17.3 |
7 (13.4) |
37 (52.1 |
15 (21.1 |
23 (41%) |
10 (17.8 |
4 (7.1%) |
44 (78.5 |
12 (21.4 |
| Ceftriaxone | 20 (31.7) |
11 (17.4) |
10 (15.8) |
39 (61.9 |
24 (38.1 |
23 (39.6 |
10 (17.2) |
4 (6.9) |
44 (75.8) |
14 (24.1) |
| Imipenem | 2 (18.2) |
2 (18.2) |
6 (54.5) |
10 (90.9 | 1 (9.1) |
2 (50) |
0 | 2 (50) |
4 (100) |
0 |
| Nitrofurantoin | 5 (6.9) |
0 | 0 | 3 (7.1) |
1 (1.4) |
6 (9.5) |
0 | 0 | 4 | 0 |
a:ESBL producer, b: ESBL non producer
Quinolone resistance, MDR, ESBL production and Integrons carriage
Of 135 isolates, 97 (71.8%) isolates presented with integrons, while in others neither integrase genes of class 1 and 2 (intI1, intI2) nor 3 (intI3) was observed.
Integrase genes were carried by 62 (72%) bacteria producing ESBL, including 27 (65%) E. coli and 35 (77.7%) K. pneumoniae. Presence of class 1 integrons in E.coli was observed to be associated with the resistance of the isolates to ceftriaxone, ceftazidime, gentamicin and nalidixic acid while, class 2 integron presence was related to the non- susceptibility of isolates to imipenem, nalidixic acid and co-trimoxazole (Table 2). In contrast, presence of class 1 integrons in K.pneumoniae was associated with resistance towards imipenem, nalidixic acid, ceftazidime and gentamicin, while the resistance to gentamicin and co-trimoxazole was observed to be associated with the presence of class 2 integrons (Table 2), compared with 10 (22.2%) ESBL-negative isolates (p<0.05). Ciprofloxacin resistance (MIC ≥4 µg/ml) in both E.coli and K.pneumoniae was related significantly (χ2= 8.8; p< 0.01) with the presence of integron class 1 and co-presence of integron class 1 and 2. This resistance was also significantly (χ2= 14.983;p< 0.001) related to the presence of ESBL in isolates as compared to non ESBL production. On the other hand, nalidixic acid resistance in both E.coli and K.pneumoniae was related significantly (χ2= 8.2; p< 0.01) with the presence of only class 1 integron. This resistance was also significantly (χ2= 16.625; p< 0.001) related to the presence of ESBL as compared to non ESBL production.
The association existed between presence of integrons and drug resistance to cefamandole, ceftriaxone, ceftazidime, gentamicin, co-trimoxazole, nalidixic acid and ciprofloxacin (Table 2).
Class 1 (n= 43; 40.5%) and 2 (n= 21; 19.8%) integrons were found in 78 (73.5%) of 106 MDR isolates. Fourteen (13.2%) MDR isolates possessed both intI1 and intI2 genes.
In respective to the organism, 21(35.6%) quinolone resistant and 11 (18.6%) MDR E.coli had intI1 and intI2 genes respectively. Nine (15.2%) E.coli harbored both intI1 and intI2 integrase genes. In comparison, 24 (51%) quinolone resistant and 11(23%) MDR K.pneumoniae isolates possessed intI1 and intI2 respectively and 5(10.6%) isolates had both intI1 and intI2 (Table 3). Class 3 integron was not found in any of the tested bacterial species.
Table 3. Association of ESBL production, presence of integrase genes and MDR with quinolone resistance in E.coli and K.pneumoniae clinical isolates .
| Ciprofloxacin resistance a | Nalidixic acid resistance a | ||||
| E.coli | K.pneumoniae | E.coli | K.pneumoniae | ||
| ESBL producer | 74.2a (26 /35)b | 29 (11/38) | 91.4 (32/35) | 47.3 (18/38) | |
| ESBL non producer | 69.2 (9/13) | 18.2 (2/11) | 92.3 (12/13) | 72.8 (8/11) | |
|
INTEGRON class |
1 | 81.8 (9/11) | 27.2 (6/22) | 100 (11/11) | 50 (11/22) |
| 2 | 63.6 (7/11) | 20 (1/5) | 81.8 (9/11) | 80 (4/5) | |
| 1,2 | 100 (9/9) | 25 (1/4) | 100 (9/9) | 50 (2/4) | |
| INTEGRON not detected | 58.9 (10/17) | 27.7 (5/18) | 88.2 (15/17) | 50 (9/18) | |
| MDR | 69.2 (27/39) | 29.7 (11/37) | 64.8 (37/39) | 54 (20/ 37) | |
| Non MDR | 88.8 (8/9) | 16.6 (2/12) | 22 (2/9) | 50 (6/12) | |
a Percentage of isolates
b Number of isolates/total number of quinolone resistant isolates
In relation to harboring integrase genes, class 1 integrase gene was being possessed by 22 K.pneumoniae and 11 E.coli. In contrast, class 2 integrase gene (intI2) was possessed more frequently by E.coli (n=11) isolates over K.pneumoniae (n=5) and this association was significant (p<0.05).
Discussion
Our study analyzed 234 E.coli and K.pneumonaie isolates obtained from various clinical specimens from inpatients and outpatients, comprehensively including community and hospital associated infections for quinolone resistance, ESBL production, multidrug resistance, possession of integrase genes and the association between them.
The microbial etiology of urinary tract infections (UTI) has been well-established and reasonably consistent. Escherichia coli remains the predominant uropathogen (80%) isolated in acute community-acquired uncomplicated infections, followed by gram positive and other gram negative organisms.25 In our study, E.coli was the predominant (82.4%) cause of community acquired UTIs, though 65.15% hospital associated UTIs were also due to this organism. We found K.pneumoniae to be principle cause of bloodstream infections. Similar reports are available which supports this finding.26-28 Though these two bacteria are leading cause of clinical infections in hospital as well as community based patients,9 however, increasing trend of antimicrobial resistance is a serious concern which has tempered the therapeutic options. Forty five (31.91%) of E.coli (34/110; 30.9%) and K.pneumoniae (11/31; 35.4%) obtained from urine specimens were resistant to nalidixic acid and those (46.55%) recovered from other clinical specimens were resistant to ciprofloxacin. Several reports are available on the mechanism of quinolone resistance in either E.coli or K.pneumoniae or even both or quinolone resistance in ESBL producing bacteria; however, no report is available on the actual resistance of nalidixic acid and ciprofloxacin in these two isolates 29-35, except few published studies on urinary isolates,36-40 whereby low prevalence has been reported as compared to our study. Study performed in our neighboring country reported 17% and 38% of E. coli isolates obtained from uncomplicated and complicated UTI respectively, were found resistant to ciprofloxacin.40
Of 72 quinolone resistant E.coli, and 63 K.pneumoniae isolates, high resistance to 3rd generation cephalosporins was observed, non-susceptibility being in the range of 49.2% - 85.9% and 52.3%- 96.8% respectively, which is quite high. The importance of infections due to ESBL producing E.coli and Klebsiella species has been increasingly recognized in recent years.41 A significant increase in the prevalence of fluoroquinolone resistance (p<0.001) was evident in their study conducted among the ESBL-E.coli and K.pneumoniae isolates over the 5 year study period. Another significant feature of their case – control report is fulfilling the criteria of MDR by 18.8% isolates. The only independent risk factor for MDR ESBL- E.coli and K.pneumoniae was infection with K. pneumoniae. Schwaber et al.42 noted high levels of co-resistance (≥40%) among their isolates for all agents except amikacin and imipenem. Another research study analyzed 867 non-repeat isolates comprising 8 species, originating from the community and 23 European hospitals, and showed a significant relation between MDR and the presence of integrase genes, independent of species or origin.13 Our study was in concordance with this research study which found 75.6% of their isolates as ciprofloxacin resistant and integron positive. We found 74.6% K.pneumoniae and 81.9% E.coli to be simultaneously resistant to other antibiotics appearing as multi drug resistant isolates, which suggest for limitations and precise use of antibiotics in our region. The present study showed the presence of class 1 and 2 integrons in 73.5% of MDR isolates and 13.2% of them possessed both intI1 and intI2 simultaneously. Our isolates had comparatively low imipenem resistance. Phenotypic resistance pattern disclosing cephalosporin resistance with aminoglycoside and imipenem phenotype was disclosed by 6.6% isolates.
Ciprofloxacin resistance was related significantly with the presence of integron class 1 and co-presence of integron class 1 and 2 together in our isolates. This resistance was also significantly related to the presence of ESBL producing isolates as compared to non ESBL production. Nalidixic acid resistance was related significantly with the presence of only class 1 integron in the isolates studied. This resistance was also significantly related to the presence of ESBL production. Our study had various limitations, with major one being study of risk factors for such a high resistance. Though we did not perform this, nevertheless we can assume exposure to antibiotics as one of major predisposing factor as our University based tertiary hospital is a core center for all North West region and patients first treated at their primary care center are referred for further treatment. Another factor may be misuse of antibiotics as antibiotics are available over- the- counter. In UK, nursing home residents had very high prevalence of gut carriage of MDR E. coli.43 Exposure to antibiotics was high among residents, with carriers having spent significantly more days receiving trimethoprim or FQs in their published report. The presence of an ESBL determinant significantly curtails the number of antimicrobial agents, and limits therapeutic option. In addition, frequent links between ESBL genes and other resistance genes on the mobile DNA elements that are involved in their dissemination, ESBL producers often present as complex multidrug resistant phenotypes.44-46
Conclusion
Prevalence of quinolone resistance in E.coli and K.pneumoniae in our clinical setting has contributed to augmentation in antibacterial resistance. High resistance to cephalosporins in ESBL-producing pathogens restricts the possibilities for effective treatment of infections. Appropriate infection control measures should be aimed at minimizing the spread of ESBLs and this should be specified as a high priority. A hopeful prospect in our study was low resistance to carbapenem.
Prevalence of class I and II integron in our quinolone resistant isolates is a similar trend observed in other published studies, however, co- prevalence with ESBL and multi drug resistance is a striking feature of our study. This is a therapeutic concern and requires further investigation taking into account the associated risk factors and study of gene cassettes. Presence of integrons warns dissemination of antibiotic resistance. Additionally, in view of confirmation of high resistance towards nalidixic acid and FQ by MICs, it is necessary that these tests should be available in the hospital for exact detection of antibiotic resistance.
Acknowledgments
This work was supported by grant from the Research Center of Infectious diseases and Tropical Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran (Grant No.91-17). This manuscript is part of MSc thesis of first author (Thesis No.91/2-3/5).
Ethical Issues
Not applicable.
Conflict of Interest
No potential conflicts of interest.
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