In this study, we determined the frequency and mode of transfer of resistance to antimicrobial agents by conjugation, among a number of multi-drug resistant Enterobacteriaceae isolates. These are all ESBL producing and/or fluoroquinolone and aminoglycoside resistant. Most of these isolates harbor the pC15-1a plasmid, that carries the bla
gene in addition to the aminoglycoside modifying enzyme aac(6')lb-cr gene which also confers some resistance to the fluoroquinolone ciprofloxacin as demonstrated in a previous study . The MDR region is  indeed harbored on the pC151a or the pCTX15 plasmids since first, the plasmids characterization done in the previous study showed that the hpa1 digestion profile was related to the pC151a or the pCTX15 plasmids and second the PCR based detection of the bla CTX-M-15 and the aac6'lb-cr genes on transferable plasmids in transconjugants were on pC15-1a or the pCTX-15 plasmids.
Molecular epidemiology analysis confirmed that conjugative isolates are not clonal and transfer of antimicrobial resistance was acquired through horizontal transfer of plasmids. Besides conjugative strains were linked epidemiologically to multiple sources. No single conjugative strain harboring the plasmid encoded antimicrobial resistant genes was circulating in the medical center.
Conjugation experiments in this study have shown that about 50 percent of the Enterobacteriaceae isolates were able to transfer resistance to cefotaxime, which is a significantly high prevalence rate of transfer of resistance among ESBL producing multi-drug resistant isolates. As demonstrated, conjugative isolates harbored the plasmid encoded tra genes encoding the transferase proteins TraM responsible for mating aggregation, TraY directing the nicking enzyme TraI to OriT,  TraI inducing nicking and unwinding  and TraD involved in pumping the DNA into the recipient cells  and hence were able to transfer resistance.
All the isolates were resistant to the 3rd generation cephalosporins, and conjugative ones were able to transfer resistance against these antimicrobial agents to the recipients. Most of the isolates were able to transfer high-level resistance against cefotaxime and cefepodoxime to the transconjugants which revealed MIC equal or above 256. It was found however, that the MIC of ceftazidime for most of the donor isolates was less than that usually reported for CTX-M-15 producing isolates (between 128 and 256 μg/ml) (1). CTX-M-15 differs from its relative CTX-M-3 by a single amino-acid substitution, which increases its activity against ceftazidime. Most of the bla¬
genes encountered in ESBL- producing isolates are located on plasmids downstream an ISEcp1 insertion sequence which harbors the -10 and -35 promoter sequences (TTGACA and TAAACT respectively) essential for the high expression of the gene and hence, the increased activity against ceftazidime and other third generation cephalosporins. These were detected in a previous study . However, CTX-M-15-producing strains with low activity against ceftazidime were previously reported in many countries including UK and the United Arab Emirates [5, 8]. A study that was done by Wooford et. al on the UK isolates, showed that there is a number of CTX-M-15-producing E. coli for which ceftazidime had MIC values equal or less than 32 μg/ml, which is lower than the usually reported for the CTX-M-15 producing Enterobacteriaceae (between 128 and 256 μg/ml) . Genotypic characterization by Woodford et al. demonstrated that the bla
gene was located on a plasmid downstream the ISEcp1; however, an IS26 insertion element was present within the terminal inverted repeat of ISEcp1, separating bla
from its usual promoter located in the ISEcp1. This genotypic characteristic was thought to be responsible for the lower bla
expression, and hence, the decreased ceftazidime MIC for the UK and the UAE strains. We suspect that the same genotypic feature is also responsible for the level of resistance against ceftazidime that is lower than expected in our CTX-M-15 producing strains.
All the transconjugants on the other hand, had intermediate resistance to ceftazidime. The seven isolates that were resistant to ceftazidime with MIC above 96 were unable to transfer this resistance to the recipients, for which the MIC of ceftazidime did not cross 32 μg/ml. This could have resulted from the presence of chromosomally-mediated β-lactamase genes in the parental strains that could not be transferred to the recipients.
With respect to the transfer of resistance to ciprofloxacin, it was noticed that all except one of the transconjugants revealed low-level resistance, resulting from the transfer of aac(6')-lb-cr. This gene has an aminoglycoside-modifying activity, and crossed the antimicrobial agents' class boundaries by its activity also against the fluoroquinolones. Although the degree of resistance conferred is small, this gene was shown to act additively with other genes that confer resistance against fluoroquinolones, like the plasmid-mediated qnr genes, or chromosomal mutations. Despite the relatively low activity of the aac(6')-lb-cr gene product against fluoroquinolones, it could play an important role in facilitating the selection of strains with mutations in the chromosomal gyrA, gyrB, and parC genes, among a bacterial population that is exposed to be fluoroquinolone resistant.
The qnr genes are other important plasmid-mediated genes that confer resistance to fluoroquinolones. qnrA is the first discovered and is found worldwide. qnrB and qnrS are recently emerging, and are still not as widespread . Only one of our isolates, E. coli 1, was able to transfer intermediate-level resistance against ciprofloxacin to transconjugants, with an MIC in the transconjugant of 3 μg/ml; whereas in all the other transconjugants, ciprofloxacin had a maximum MIC value of 0.125 μg/ml. We suspected the presence of one of the plasmid-mediated qnr genes in E. coli 1 in addition to the aac(6')-lb-cr, and indeed, PCR amplification on the plasmid extract from this isolate confirmed the presence of both genes in the donor and the transconjugant. The higher MIC of ciprofloxacin in the E. coli 1 transconjugant compared to that of the transconjugants of the other isolates, leading to intermediate-level ciprofloxacin resistance is most probably due to the simultaneous transfer of both the qnrS and aac(6')-lb-cr genes to them, and hence, the coordinating action of 2 different fluoroquinolone-resistance mechanisms. It is known that the qnrS gene by itself does not confer intermediate-level resistance to ciprofloxacin [21, 22].
In conclusion 49% of the E. coli and K. pneumoniae of non-clonal conjugative isolates in our medical center were able to transfer resistance to third generation cephalosporins in transconjugants as encoded by the bla
gene along with its promoter in the ISEcp1 insertion element. In addition the isolates were also able to transfer a low-level resistance to ciprofloxacin and variable resistance to aminoglycosides, encoded by aac(6')-lb-cr gene. Conjugation occurred in isolates expressing the tra encoding transferase genes. Multiple conjugative strains harboring the plasmid encoded antimicrobial resistant genes were circulating in the medical center.