Clonal relation and antimicrobial resistance pattern of extended-spectrum β-lactamase-and AmpC β-lactamase-producing Enterobacter spp . isolated from different clinical samples in Tehran , Iran

Introduction: Here, we determined the genes encoding antibiotic resistance enzymes and virulence factors and evaluated the genetic relationship between Enterobacter spp. isolated from different clinical samples. Methods: A total of 57 clinical isolates of Enterobacter spp. were tested for the production of extended-spectrum β-lactamases (ESBLs), carbapenemase, and AmpC using phenotypic and genotypic methods. Results: The most common ESBLs and AmpC β-lactamases were blaTEM (63.3%) and blaEBC (57.7%), respectively. The most prevalent virulence gene was rpos (87.7%). The random amplified polymorphic DNA (RAPD) patterns of strains were genetically unrelated. Conclusions: RAPD polymerase chain reaction analysis revealed high genetic diversity among isolates.

Enterobacter species may cause severe nosocomial infections, including bloodstream, respiratory tract, and central nervous system infections as well as endocarditis 1,2 .Nosocomial infections caused by these microorganisms have been associated with high rates of mortality and morbidity 1 .Enterobacter cloacae and Enterobacter aerogenes are the most common species isolated from clinical samples 3 .Several virulence genes are involved in the pathogenesis of these microorganisms [4][5][6][7] .Curli fimbria, encoded by csgBAC, is an important factor for cell adhesion, aggregation, and biofilm formation in many enterobacteria 4 .In addition, RpoS regulation is known to play an important role in multiple stress conditions such as acid, heat, and oxidative stress, starvation, high osmolarity, and near UV exposure 5 .Another important virulence factor is the type III secretion system encoded by FliI that delivers a variety of effectors directly into the cytosol of host as well as aerobactin, encoded by the iutA, described as a virulence factor related to iron acquisition from host-binding proteins 6,7 .β-lactam antibiotics, especially third-generation cephalosporins and carbapenems, are used to treat infections cause by several species of Enterobacter [1][2][3] .β-lactamase enzymes, including extended-spectrum β-lactamases (ESBLs) and AmpC, are involved in the mechanism underlying resistance to β-lactam antibiotics in Enterobacter spp [1][2][3] .ESBLs are often encoded by genes located on large plasmids that also carry genes for resistance to other antimicrobial agents such as aminoglycosides and fluoroquinolones 1 .ESBLs are capable of hydrolyzing penicillins, broad-spectrum cephalosporins, and aztreonam, but may not hydrolyze cephamycin, and are inhibited by clavulanic acid.AmpC β-lactamases are usually encoded on the bacterial chromosome and in some cases on the bacterial plasmid (plasmid-mediated AmpC) 3 .In Iran, ESBL production was recently reported in 44.28% of E. cloacae isolates 1 .Despite the high incidence of Enterobacter spp.infection among Iranian patients, very little is known about the antibiotic resistance pattern, virulence factors, and molecular characteristics of Enterobacter spp.isolates.In the current study, the genes encoding antibiotic resistance enzymes and virulence factors were determined and the genetic relationship between Enterobacter spp.isolated from different clinical samples was evaluated.

Bacterial isolates
A total of 57 isolates of Enterobacter spp.were obtained from different patients admitted to three teaching hospitals of the Tehran University of Medical Sciences between September 2013 and April 2014.The isolates were collected from various clinical samples, including urine, wounds, tracheal aspirate, and blood.No duplicate isolates from the same patient and no environmental strains were included in this study.All isolates of Enterobacter spp.were identified by standard biochemical tests 8 .

Random amplified polymorphic DNA-PCR
For molecular analysis of isolates, random amplified polymorphic DNA (RAPD)-PCR was performed as previously described 12 .In brief, PCR protocol comprised a pre-denaturation step at 95 °C for 5 min, followed by 30 cycles of 60 s at 95 °C, 60 s at 33 °C, and 60 s at 72 °C.A final extension step was performed at 72 °C for 10 min.PCR products were separated by electrophoresis on 1% agarose gels with 0.5× Tris-borateethylenediaminetetraacetic acid (EDTA) buffer (TBE buffer).Gels were stained with ethidium bromide and the images were captured using a gel documentation system.Isolates that differed by more than two prominent bands were assigned to different types.
Our study revealed that 35.1%, 12.3%, and 10.5% of isolates were resistant to imipenem, levofloxacin, and gatifloxacin, respectively.Previous reports from Iran have shown that the resistance rate of Enterobacter isolates to imipenem and gatifloxacin was 2% and 7%, respectively 1 .Our results indicated the significant increase in the resistance to carbapenem and ciprofloxacin, which may be attributed to the inappropriate and widespread use of antibiotics 1 .Of the 30 isolates that were recognized as phenotypically positive for ESBL production in this study, 27 were positive for ESBL genotypes.In the study conducted by Kanamori et    was the most common type of AmpC β-lactamase, followed by bla ACC (16.6%).Miró et al. reported that the CMY (78.3%) and DHA (19.5%) families were the most prevalent type of AmpC β-lactamase in 35 hospitals in Spain 13 .However, the prevalence of ESBL and AmpC-producing Enterobacter spp.varied among different studies, which may be associated with the differences in the geographical area, type of infection, and settings (hospital or community).Similar to previous reports, we observed the coexistence of ESBL-encoding genes in clinical isolates 1,2 .Several virulence factors have been identified in the pathogenesis of Enterobacter spp [4][5][6][7] .The majority of isolates (87.7%) carried rpos and fliI.The high frequency of these genes may indicate that these genes are essential for the development of disease.In contrast to the findings of our study, Krzyminska et al. observed that only 27% of isolates harbored fliI (TTSS gene) 6 .In the current study, the frequency of csgB, csgD, and csgA was 70.2%, 68.4%, and 59.6%, respectively, which is lower than that reported in the previous study by Akbari et al.These authors showed that csgD and csgA genes were present in 100% and 77.75% of isolates, respectively 14 .The genes iutA and fepA were found in 54.4% and 50.9% of isolates in our study.Mokracka et al. reported that 49% of E. cloacae strains produced aerobactin 15 .However, differences were observed in the frequency of virulence genes reported in different studies; this difference may be associated with the variation in the geographical area, clinical samples, and other factors.RAPD-PCR analysis revealed the significant genetic heterogeneity.In addition, molecular analysis demonstrated that more than 90% (28/30) of ESBL-producing isolates were clonally unrelated, indicating that the reported infections had no relation with clonal outbreak.In conclusion, bla TEM , bla CTX-M , and bla EBC are the most common resistance gene types and more than 50% of isolates harbored virulence genes.RAPD-PCR analysis revealed high genetic diversity among isolates.
al. from Japan, 22 of 364 Enterobacter spp.were identified phenotypically positive for ESBL production, but only 11 isolates harbored ESBL genes; ESBL genes were undetected in the remaining 11 isolates 2 .Discrepancy between disc tests and PCR detection results may be associated with the lack of any standardized method

TABLE 1 :
The oligonucleotide primers used in this study for the amplification of virulence genes.

TABLE 2 :
2ontinuation.for the detection of ESBLs in Enterobacter spp2.In the present survey, 30 (52.6%) Enterobacter isolates were found to be ESBL producers.Kanamori et al. also reported that 6% Enterobacter spp.wereESBL producers 2 .The high prevalence of ESBL-positive isolates in our study may be associated with the extensive use of third-generation cephalosporins for the treatment of Enterobacter infections.It should be noted that 10% (3/30) isolates were ESBL negative and eight isolates that were recognized phenotypically positive for carbapenemase failed to show any carbapenemase-related genes, suggestive of the involvement of other resistance mechanisms.In our study, 26.7% (8/30) of ESBL-positive isolates were MDR.Peymani et al. reported that all ESBL-positive Enterobacter isolates were MDR1.In our study, bla TEM and bla CTX-M were the most common ESBL resistance genes, which were frequently reported in other countries2.In the present study, bla EBC (57.7%)