Extended-Spectrum β-Lactamases among Enterobacteriaceae Isolated from Urinary Tract Infections in Gaza Strip, Palestine

Background Extended-spectrum β-lactamase-producing organisms causing urinary tract infections are increasing in incidence and pose a major impendence to health-care facility, having limited therapeutic options. This study aimed to assess the prevalence of ESBLs in Enterobacteriaceae isolates causing urinary tract infections in Gaza strip, Palestine, and to characterize β-lactamase types and associated resistance genes. Methods Eighty-five Enterobacteriaceae isolates were recovered from urinary tract infections within three months in Gaza Strip hospitals. The characterization of β-lactamase genes and the genetic environments of CTX-M, the identification of associated resistance genes, and the presence and characterization of integrons were tested by PCR and sequencing. Results The occurrence rate of ESBL among tested isolates was 30 (35.3%), and among ESBL-positive isolates, blaCTX-M was the highest followed by blaTEM. ESBL-CTX-M-1 group was confirmed in 93.3%, and the remaining carried CTX-M-9 group. CTX-M-15, CTX-M-3, CTX-M-1, CTX-M-14, CTX-M-27, and CTX-M-37 enzymes were demonstrated among the isolates with the majority (73%) being CTX-M-15. ISEcp-1 was demonstrated in 27 (90%, high incidence) of ESBL isolates. Class 1 integrons have been detected in higher rates (53.3%) in ESBL-positive isolates in comparison with non-ESBL isolates (6, 33.3%). Cassettes of integron-1 contain (aadA1, aadA2, aadA5, dfrA5, dfrA7, dfrA12, and dfrA17) genes. The aac(6′)-Ib-cr gene was demonstrated in 36.7% of ESBL-positive isolates. Conclusions This study indicates that blaCTX-M-15 was the most prevalent β-lactamase in this region. Our study demonstrates for the first time in Palestine the identification of blaCTX-M-15 in P. rettgeri and S. liquefaciens, also blaCTX-M-37 in E. cloacae. The coexpression of multiple β-lactamase genes with aac(6′)-Ib-cr and qnr in the presence of ISEcp-1 and integrons in individual strains will increase the dissemination of highly resistant strains. ESBL producers were more resistant than non-ESBLs producers for almost all tested antibiotics.


Introduction
Urinary tract infection (UTI) is one of the most common widespread infections, mainly caused by Enterobacteriaceae, especially Escherichia coli, that are encountered by hospitalized and outpatients [1]. Normally, UTIs are treated with different classes of antibiotics such as β-lactams, β-lactam/ β-lactamase inhibitors, carbapenems, and fluoroquinolones [2]. However, recent data worldwide reveal that these uropathogens have become resistant to most conventional drugs [3].
Extended-spectrum β-lactamases (ESBLs) are bacterial enzymes that hydrolyze oxyimino-cephalosporins and confer resistance to broad-spectrum cephalosporins and aztreonam [4]. Enterobacteriaceae harboring ESBLs is a global problem with limited available treatment options [5]. ESBL-producing bacteria are related with infections that are consequences of bad clinical facilities, inappropriate antibacterial therapy, prolonged hospital stays, and greater hospital costs [6]. In the past years, there has been an increase in widespread dissemination of β-lactamase-mediated resistance with high significance in the prevalence of ESBLproducing Enterobacteriaceae [7].
Early investigation of patients infected with ESBL-producing urinary pathogens is necessary to prescribe the most efficient therapy and minimize the dissemination of infection by applying preventive measures. erefore, the aim of this study was to detect the occurrence of ESBL-producing Enterobacteriaceae and investigate the molecular characteristics of β-lactamase-producing isolates obtained from three hospitals in Gaza Strip, Palestine.

Isolation and Identification.
Eighty-five Enterobacteriaceae isolates were recovered from urinary tract infection samples obtained in various units of three hospitals in Gaza Strip, Palestine, during the period of three months in 2013. Only one bacterial isolate per patient was included in this study. All collected specimens were sent to the microbiology laboratory to be processed for bacterial isolation and identification. Standard methods for isolation and identification were used [17]. e isolates were confirmed to genus and species level by amplification and sequencing of 16S rRNA gene.

Identification of β-Lactamase Genes and Genetic
Environment of bla CTX-M Genes. PCR amplification and sequencing were used to determine the presence of the genes encoding TEM, SHV, OXA, and CTX-M type β-lactamases. e genetic environment surrounding the bla CTX-M genes was studied by the amplification of ISEcp-1, orf477, and IS903 [19].

Detection and Characterization of Integrons.
e presence of intI1 and intI2 genes encoding for classes 1 and 2 integrase, respectively, and the presence of qacED1-sul1 genes within 3′-conserved region of class 1 integrons was identified by PCR. e variable regions of class 1 and 2 integrons were studied in intI1-or intI2-positive isolates by PCR and sequencing to identify the gene cassettes [19].
Positive and negative controls were kindly provided from the Universite´Tunis-El Manar, Tunis, and were used in all PCR and sequencing experiments.

Data Analysis.
e data of antimicrobial resistance of non-ESBL and ESBLs of Enterobacteriaceae isolates were analyzed by SPSS version 20 software (IBM Corporation, Somers, NY) by applying a Pearson's chi-square test. Level of statistical significance was set at P < 0.05.

Bacterial Strains.
In this study, we screened 85 Enterobacteriaceae isolates from urine from three Palestinian hospitals in Gaza Strip for the determination of β -lactamase-encoding genes and to characterize their type, genetic environments of CTX-M, and associated resistance genes.
On comparing the antibiotic resistance of ESBL and non-ESBL Enterobacteriaceae, both showed low resistance to imipenem in comparison to other tested antibiotics. However, resistance to other antibiotics was higher among ESBL than non-ESBL isolates with statistical significance (Table 1). To the end, the resistant pattern of ESBL producers was significantly higher than that of non-ESBL producers for all 15 tested antibiotics except for imipenem (Table 1).
e SHV-encoding genes were found among five isolates of K. pneumoniae in two variants; bla SHV-1 and bla SHV-11 with the frequency of 2 and 3, respectively. Finally, bla OXA-1 was detected in only one isolate of E. coli (Table 2).

Genetic Environments of bla CTX-M Genes.
e genetic mechanisms that may be involved in the expression and mobilization of bla CTX-M genes and the genetic environments upstream and downstream of bla CTX-M genes were studied in ESBL-positive Enterobacteriaceae isolates by PCR and sequencing. e sequence of orf477 was found downstream of the ESBL-CTX-M-1 group members (bla CTX-M-15 , bla CTX-M-3 , bla CTX-M-1 , and bla CTX-M-27 genes) in twentyseven isolates. e insertion sequence (IS903) was identified downstream of the bla CTX-M-14 gene in one isolate (NT134), whereas the downstream region of bla CTX-M-27 and bla CTX-M-37 in E. cloacae and E. coli, respectively, was unknown. e insertion sequence (ISEcp-1) was found upstream bla CTX-M genes in twenty-seven isolates; however, the upstream region of bla CTX-M-37 , bla CTX-M-15 , and bla CTX-M-14 in NT60, NT117, and NT134 isolates, respectively, was unknown.

Integrons and Arrangement of Gene Cassettes.
Class 1 integron has been demonstrated in ten ESBL-positive E. coli isolates with the following gene cassette arrangements: dhfr17 + aadA5 (6 isolates), dhfrA7 (2 isolates), and dfrA12 + aadA2 (one isolate); three of those integrons lacked the qacEΔ1 and sul1 genes. In K. pneumoniae, class 1 integrons were present in 3 of the 5 ESBL-producing isolates with two different genetic arrangements (dfrA12 + aadA2) and dfrA5. Int1 was identified in two ESBL-positive E. cloaca, the gene cassette implicated in the resistance to streptomycin (aadA1) was detected in one isolate, and one of those integrons lacked the qacEΔ1 and sul1 genes. e cassette that conferred resistance to trimethoprim (dfrA12) and streptomycin (aadA2) was found in integron-1 among ESBLproducing S. liquefaciens.

Discussion
A total of 85 Enterobacteriaceae isolates obtained from urinary tract infections from in-and outpatient populations during a three-month time period were evaluated for the production of ESBL, β-lactamase enzymes, and associated resistance genes. e finding showed that among 85 urine isolates, E. coli was the most prevalent representing 60 (70.6%) of isolates followed by K. pneumoniae 15 (17.6%). is is in agreement BioMed Research International with other studies investigating Enterobacteriaceae causing urinary tract infections in Sri Lanka and Qatar [5,21]. e prevalence of ESBL among our isolates was confirmed in 30 isolates (35.3%). is was similar to the findings of Liu et al., Giwa et al., and Caccamo et al. [22][23][24]where ESBL production among Enterobacteriaceae causing urinary tract infections was found to be 33.4%, 34.3%, and 36.0%, respectively.
Our findings demonstrated that imipenem was the most effective drug against ESBL isolates. Studies performed in Palestine from different clinical awards demonstrated the same as our findings about the effectiveness of imipenem [1,[25][26][27].
e results of our study showed that the frequency of antibiotic resistance among ESBL producers is higher than that of resistance in nonproducers; our findings correlate with other studies in Tanzania and India [28,29]. e spread of ESBL producers in hospitals and their increased antibiotic resistance is alarming.
Resistance to imipenem was found to be low among ESBL producers (20.0%) and non-ESBL producers (12.7%) without statistical significance. e lower resistance to imipenem among ESBL and non-ESBL's was reported in India [30]. e mechanism of resistance to carbapenem occurs by bacterial production of β-lactamases and decreases the permeability of the antibiotics by changes in porin channels in the cell wall or reduced susceptibility of bacterial cell toward meropenem through upregulation of efflux pumps [31]. e imipenem resistance in our study could be due to production of carbapenemase. is may be because patients in Palestinian hospitals are treated with carbapenems which may have a role in development of multidrugresistant strains. Detection of carbapenemase genes are beyond the aims of this study and will be our planned future work.
Molecular genotyping of ESBL-containing isolates revealed the highest presence of CTX-M genes (100%) followed by TEM genes, which is in agreement with previous reports from the region and around the globe [1,32]. CTX-M enzymes have become dominant extended-spectrum β-lactamases in Europe [33] and in many Middle Eastern countries [34].
In this report, 93. is finding is in agreement with study in Qatar that showed among the ESBL-producing Enterobacteriaceae infections in urine 90% of the CTX-M belonged to CTX-M-1 group and 8% belonged to the CTX-M-9 group [5]. In a recent study in USA, among the ESBL-producing Enterobacteriaceae, 80% and 20% were positive for CTX-M-1 group and CTX-M-9 group, respectively [32].
Genotypic characterization of all ESBL-positive isolates revealed that the majority (73%) of the CTX-M type was CTX-M-15. A lot of reports worldwide described that CTX-M-15 is the most prominent distributed ESBL-CTX-M enzyme [14][15][16]. Rapid dissemination of CTX-M-15 enzyme is reported in many countries. e easy transfer of this gene is associated with the epidemic plasmid [35].
One of the significant findings of this study is that this is the first report of bla CTX-M-15 in P. rettgeri and S. liquefaciens in the Middle East and also the first report of bla CTX-M-15 in E. cloacae in Palestine. In the previous reports, bla CTX-M-15 was identified in P. rettgeri causing urinary tract infections in Croatia [36], and bla CTX-M-15 was reported in E. cloacae in Southern China [22], Egypt [11], and Yemen [37].
In         [39]; this enzyme was described in clinical isolates of Enterobacteriaceae in Korea and France [40,41]. Sequencing the amplification of PCR products from the two E. coli isolates detected the CTX-M-encoding gene as bla CTX-M-1 ; this gene has been demonstrated in clinical Enterobacteriaceae in France and Italy [41,42]. CTX-M-27 enzyme has been reported in nosocomial outbreaks caused by Salmonella enterica in a neonatal unit in Tunisia [43]. Our study reported for the first time the presence of CTX-M-37 in clinical Enterobacteriaceae isolates in the Middle East. Its presence was recently reported in clinical strains of E. cloacae in Mongolia [44] and was also identified in S. enterica serotype Isangi from South Africa [45].
Our results showed that ten bla CTX-M-15 -containing isolates carried more than one β-lactamase gene, and three K. pneumoniae isolates harbored bla CTX-M-15 , bla SHV-1 , and bla TEM-1 . One isolate coexpressed bla CTX-M-15 with other β-lactamase gene, and E. cloacae carried bla CTX-M-15 , bla TEM-1 , and bla OXA-1 . In previous studies, Enterobacteriaceae isolates carrying multiple β-lactamase genes have been reported [34,46,47]. e occurrence of multiple β-lactamase genes in individual strains is a concern because it will enhance coresistance and greater resistance to various classes of antibiotics.
Insertion sequences and integrons have played an important role in the dissemination of bla CTX-M . ISEcp-1 is the most common insertion element associated with bla CTX-M . Moreover, the role of ISEcp-1 is mobilization of the bla CTX-M gene, and it acts as a strong promoter which enhances the expression of bla CTX-M genes [48]. ISEcp-1 was demonstrated in high incidence (90%) among our isolates which is a growing problem of high resistance in our hospitals that can carry high risk through dissemination of bla CTX-M between patients. ISEcp-1 insertion sequence was found upstream of CTX-M enzyme in Enterobacteriaceae isolates from Tunisia and Croatia [19,36].
In the present study, class 1 integrons were detected and included resistance genes in the variable region encoding resistance to streptomycin (aadA1, aadA2, and aadA5) and trimethoprim (dfrA5, dfrA7, dfrA12, and dfrA17) which increases the risk of the dissemination of antimicrobial resistance by horizontal transmission by integrons. e integrons lacking qacEΔ1 and sul1 genes have been previously reported in other countries [19,49].
ESBL-positive isolates have been demonstrated to carry higher rates (16; 53.3%) of class 1 integrons compared to non-ESBL isolates (6; 33.3%). e integron contribution to transfer extended-spectrum cephalosporin resistance has been established [50]. e frequency of occurrence of integrons among our ESBL-positive isolates was 53% which is less than the rate of isolates from Europe [51]; in contrast to this, the integron frequency was 73% and 92% in isolates from Australia and India [50,52]. In our study, the percentage of integron in non-ESBL-producing isolates was almost similar to the results obtained from studies performed in Spain and India [52,53]. May be there are possibilities of transferring the integrons carrying ESBL genes and other drug-resistant determinants from ESBL to non-ESBL-producing isolates that make non-ESBL isolates more resistant and complicate antibiotic resistance problems in our hospitals. e non-β-lactam antibiotic resistance pattern of the isolates showed that the sul and aac(3)-II genes were the most prominent identified in our isolates. is is in agreement with the findings of a study conducted in Spain [54]. e variant aac(6′)-Ib-cr gene has the ability to reduce susceptibility to aminoglycosides and ciprofloxacin [16]. is gene prevalence rate in our study was 36% among ESBL-producing Enterobacteriaceae isolates in comparison with 25% in a previous study in Palestine [55]. A significant association between resistance to broad-spectrum cephalosporins and resistance to quinolones was reported [56]. e association of qnrs1 gene with bla CTX-M identified in this study was similar to the findings of a study in Algeria [57]. We also observed the associations between qnrB1 and bla CTX-M which is in agreement with previously reported results in Tunisia [58]. Moreover, the correlation between qnrA and bla CTX-M demonstrated here was also reported in Yemen [37]. e presence of β-lactamases in association with other resistance genes, qnr gene and aac(6′)-Ib-cr, in the same strain could complicate the treatment of these pathogens. ese results support previous reports that suggest larger dissemination of aac(6′)-Ib-cr with qnr determinants, in ESBL-producing isolates harboring bla CTX-M-15 gene [58][59][60].
Regarding the comparison of the occurrence of nonβ-lactam genes within ESBL and non-ESBL isolates, the nonβ-lactam genes were confirmed in 83% and 33.3% in ESBL and non-ESBL isolates, respectively. ese findings could complicate the resistance problem and limit the antimicrobial drug therapy of infections caused by ESBL producers.

Conclusions
Our study highlights the high incidence of ESBL in Enterobacteriaceae recovered from urinary tract infections in Gaza hospitals, Palestine, with the increase in antibiotics resistance for most commonly used antibiotics in our hospitals except for imipenem that showed the highest activity against ESBL isolates. ESBL producers were found to be more resistant than non-ESBLs producers for almost all tested antibiotics. is study indicates that bla CTX-M-15 was the most prevalent β-lactamase in Gaza Strip hospitals. To our knowledge, this is the first report on identification of bla CTX-M-15 in P. rettgeri and S. liquefaciens and also bla CTX-M-37 in E. cloacae in the Middle East. e associations between multiple β-lactamase genes in individual strains with other resistance genes such as aac(6′)-Ib-cr and qnr in the presence of ISEcp-1 and integrons increase the dissemination of highly resistant strains and complicate the problem of clinical conditions and treatment failure in our hospitals.

Data Availability
All data used to support the findings of the study are approved and included within the article. 8 BioMed Research International

Conflicts of Interest
e authors declare that they have no conflicts of interest.