Carriage of Class 1 and 2 Integrons in Quinolone , Extended-Spectrum-β-Lactamase-Producing and Multi Drug Resistant E . coli and K . pneumoniae : High Burden of Antibiotic Resistance

2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(3), 335-342 doi: 10.15171/apb.2015.047 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


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. 35][6][7] Furthermore, emergence of multi drug resistance (MDR) and Extended-Spectrum β-Lactamase (ESBL) production in E.coli and Klebsiella pneumoniae 8 in chorus with FQ resistance has knockdown the infrastructure of therapy substantially. 9As regards to development of antibiotic resistance, the dissemination of resistance genes among bacterial strains is being debated frequently.][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. 15The prevalence of integrons 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.

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. 19,20Duplicate isolates from the same patient were excluded.These isolates were subjected to routine antibiotic susceptibility testing performed by disc agar diffusion method. 21The 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). 21Any decrease in the zone sizes for the 3rd generation cephalosporins was used presumptively as ESBLs producer, and was confirmed later by CLSI criteria. 21ATCC 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. 21Regardless 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. 21ESBL 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. 23For 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. 24Data were analyzed using the statistical package for social sciences (SPSS 18.0, IBM SPSS, New York, USA).Contingency table analysis was done by a chisquare 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.

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).

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.

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 (χ 2 = 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 (χ 2 = 8.2; p< 0.01) with the presence of only class 1 integron.This resistance was also significantly (χ 2 = 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).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.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. 25In 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.7][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/ (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][30][31][32][33][34][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. 40Of 72 quinolone resistant E.coli, and 63 K.pneumoniae isolates, high resistance to 3 rd 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. 41A significant increase in the prevalence of fluoroquinolone resistance (p<0.001) was evident in their study conducted among the ESBL-E.coliand K.pneumoniae isolates over the 5 year study period.Another significant feature of their casecontrol 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. 13Our 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. 43Exposure 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.[46]

Table 1 .
Type of clinical specimens and distribution of E.coli and K.pneumoniae Quinolone resistance and ESBLWhen 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%

Table 2 .
Association of resistance to various antimicrobial agents and presence of integrase genes in E.coli and K. pneumoniae isolates a:ESBL producer, b: ESBL non producer 31; 35.4%) obtained from urine specimens were resistant to nalidixic acid and those