Prevalence and molecular characteristics of ESBL and AmpC β -lactamase producing Enterobacteriaceae strains isolated from UTIs in Egypt

Background Infections caused by Enterobacteriaceae are mainly treated with the β-lactam antibiotics, nevertheless, the emergence of species with plasmid-borne β-lactamases has decreased the efficacy of these antibiotics. Therefore, continuing studies on the resistance pattern of different regions is important for assessment of proper antimicrobial therapy protocols. The study aimed to characterize extended-spectrum β-lactamase (ESBL) and AmpC β –lactamase (AmpC) producing Enterobacteriaceae isolated from community-acquired UTIs in Egypt. Methods Out of 705 urine samples, 440 Enterobacteriaceae isolates were investigated to detect ESBL and AmpC β -lactamases producers by phenotypic and molecular methods. Results Out of 440 Enterobacteriaceae isolates, 311 were identified as ESBL producers by phenotypic testing. ESBL genes were detected in 308 isolates. BlaCTX-M-type was the most prevalent 254 (81.6%), out of them blaCTXM-15 was the commonest (152, 48.8%) followed by blaCTX-M-1 (140, 45%), blaCTX-M-8 (72, 23.1%) and lastly blaCTX-M-2 (4, 1.3%). blaTEM gene also was detected in a high rate (189, 60.7%). Two hundred and thirty-five (75.5%) of ESBL producers harbored blaCTX-M in combination with blaTEM and/or blaSHV genes. Multiple drug resistance in the ESBL-producers was significantly (P < 0.05) higher than in non–ESBL producers. Imipenem was the most effective drug against ESBL producers. Among 35 cefoxitin resistant isolates, 18 (51.4%) identified as carrying AmpC genes by multiplex PCR. Within AmpC β -lactamase genes, DHA gene was the predominant gene (15, 42.3%). CIT and MOX genes were also present, but in a low rate (5, 14.2% and 4, 11.4%) respectively. Co-existence of multiple AmpC genes was detected exclusively in K. pneumoniae isolates. E. coli isolates harbored DHA gene only. However, FOX gene was not detected in the study isolates. Seventeen of isolates carrying AmpC genes were also positive for ESBL genes. Conclusion The study shows that the prevalence of ESBL producing Enterobacteriaceae spread in south Egypt is alarming, however AmpC β -lactamase production is not so high.


Background
Enterobacteriaceae are the most common pathogens causing urinary tract infections (UTIs) [1]. Increasing rates of antimicrobial resistance among Enterobacteriaceae strains decrease the options for empiric treatment of these infections [2]. These pathogens are the Page 2 of 9 Mohamed et al. Antimicrob Resist Infect Control (2020) 9:198 main bacteria found to be associated with extendedspectrum β-lactamase (ESBL) production [2]. Infections caused by ESBL-producing strains are considered a serious global health concern [3,4] as these infections are associated with higher morbidity and mortality rates [5]. ESBL production is a mechanism of resistance in which the beta-lactam ring of antimicrobials such as penicillins and cephalosporins is hydrolyzed [6]. Until 2000s, blaSHV and blaTEM types of ESBLs used to be the commonest ESBL genotypes found in Enterobacteriaceae strains [7]. The corresponding genes were often found on plasmids that facilitate their rapid spread between different bacterial species [8,9]. After that, blaCTX-M types were recorded as the commonest genotypes among Enterobacteriaceae strains causing human infections worldwide (particularly blaCTX-M-15) [10]. There are other variants of β-lactamases such as AmpC β -lactamase, that can mediate resistance to several antibiotics as penicillins, cephamycins (e.g., cefoxitin and cefotetan), and oxyimino-cephalosporins [11]. Resistance to broad-spectrum β-lactams mediated by ESBLs and AmpC β -lactamase enzymes has posed a great health burden [12], particularly in developing countries where the resistance rates are high. Additionally, drug use guidelines and studies on this issue are not enough in these countries [13]. Due to a lack of solid data regarding the emergence of ESBLs and AmpC β -lactamase enzymes from Egypt, particularly south Egypt, this study aimed to determine the prevalence of ESBLs and AmpC β -lactamase production in Enterobacteriaceae isolated from patients suffering from community-acquired UTIs and characterize these strains using phenotypic and genotypic assays.  [15].

Screening for ESBLs -producing strains
According to the CLSI guidelines, isolates with inhibition zone size ≤22 mm with ceftazidime (CAZ) 30 μg and ≤ 25 mm with ceftriaxone (CRO) 30 μg were suggested to be ESBL-producers and subjected to further phenotypic and genotypic examination. Double-Disc Synergy Test (DDST) was used for confirmation of ESBL production. Standard (0.5 McFarland) inoculum of the study isolates were inoculated on Mueller Hinton agar plates. Ceftazidime (CAZ) (30 μg) and ceftriaxone (CRO) 30 μg discs were applied on agar 1.5 cm away from the center of amoxicillin-clavulanic acid (AMC) (20 μg/10 μg) disc and incubated at 35 °C for 18 h. Positive result is identified when the zone of inhibition is extended towards AMC (20 μg/10 μg) disc > 5 mm [18].  9:198 Screening for AmpC β-lactamase-producing strains Strains were screened using disk diffusion method in which cefoxitin (FOX) 30 μg disc was used. Isolates showing an inhibitory zone diameter ≤ 18 mm were suspected to be AmpC β-lactamase producers [19]. Disc Approximation Assay (D Test) was also performed; a blunting in the inhibitory zone (D shaped) around the CAZ (30 μg) towards the side of one of the inducers (IPM (10 μg), FOX (30 μg), and AMC (30 μg)) is considered as positive for inducible AmpC β-lactamase production [20].

Molecular characterization of ESBLs and plasmid mediated AmpC β-lactamase genes
DNA extraction was done using QIAamp Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. All isolates that were phenotypically resistant to β-lactams were screened for ESBL genes by the polymerase chain reaction (PCR), Including blaTEM, blaSHV, blaCTX-M (1,2,8,9,15) genes. Presence of other resistance genes previously associated with plasmids encoding blaCTX-M-15 as aac(6′)-Ib-cr was screened by PCR. A multiplex PCR was used to examine the presence of plasmid-mediated AmpC genes, including; MOX, CIT, DHA, and FOX genes. Amplified products were resolved on 2% agarose gel electrophoresis and visualized under a UV transilluminator (Biometra, Germany). The primer sequences and amplification conditions are shown in Table 1. Amplified products (one sample for each gene) sequences were analyzed (Applied Biosystems, USA), according to the BLAST software of the National Library of Medicine (http://www.ncbi.nlm. nih.gov/blast ).

Statistical analysis
Statistical analysis of demographic, clinical and laboratory data of study subjects was performed using SPSS for windows version 19.0 (IBM, USA). The chi -square test was used for analyzing categorical variables. P value < 0.05 was considered statistically significant (two-tailed).

Demographic data and distribution of Enterobacteriaceae strains
A total of 440 Enterobacteriaceae strains were isolated from urine specimens of 440 patients suffering from UTI.  Fig. 2, Table 2 and (Additional file 1: Fig Fig S4). Frequency of aac(6′)-Ib-cr gene (responsible for resistance to AK and CIP) among ESBL producers was examined by PCR. A total of 165 (53%) isolates were positive aac(6′)-Ib-cr gene. The association between aac(6′)-Ib-cr gene and blaCTX-M genes was significant (p value < 0.01) ( Table 3).

Resistance pattern in ESBL genes carrying isolates and non-ESBL genes carrying isolates
The resistance rates to most of the antimicrobial agents were significantly higher in isolates carrying ESBLs genes than in isolates that don't carry ESBL genes (p value<  9:198 .05). However, the rate of resistance to cefoxitin and nitrofurantoin in the two groups did not differ significantly (p value > 0.05). (Table 4).

Discussion
Resistance of Enterobacteriaceae to third generation cephalosporins is a worldwide problem [27], which is mainly caused by ESBLs production. Production of additional β-lactamases (AmpC) also contributes to this problem, moreover, the presence of AmpC genes is often associated with multidrug resistance [10]. Previously, AmpC -β-lactamase has received less attention, but is now identified as an important cause of resistance in Enterobacteriaceae species [10]. Global spread of β-lactamases-producing strains gives a great importance to the study of these strains in community and hospitals for reassessment of the existing treatment protocols. In Egypt, multiple studies have investigated the prevalence of ESBLs among Enterobacteriaceae isolated from hospital and community acquired-UTIs [28][29][30] [31], suggesting an increasing rate of ESBLsproducing Enterobacteriaceae spread in Egypt, that may be caused by extensive use of 3rd generation cephalosporines as empiric treatment in Egypt. The prevalence of ESBL production varies according to species, geographical areas, variations in infection control programs, different patterns of empiric antibiotic regimens and even over time. Moreover, selective pressure caused by the overuse of cephalosporins in some countries leads to the emergence of increasing rates of ESBLs production [32]. The prevalence of ESBL-production among species of our study was as follows; 100, 74.6, 69.6 and 46.6% of Citrobacter spp., K. pneumoniae, E. coli, and proteus spp. respectively. These findings disagree with some previous studies in Egypt, where ESBL-production was more frequent in E. coli isolates (17% E. coli and 1.2% of non-E. coli isolates) [28] and (97% E. coli, 82.6% K. pneumoniae and 82% Proteus) [33]. However, our finding was comparable with several studies from other African countries, that analyzed ESBL producing-Enterobacteriaceae isolated from different clinical samples. The prevalence in Uganda was 64.9% (72.7% K. pneumoniae and 58.1% E. coli) [34], in Burkina Faso was 58% (62.7% K. pneumoniae and 58.7% E. coli [35], and in Ethiopia 50.7% (52.2% E. coli and78.6% K. pneumoniae) [36]. However, our prevalence was higher than those found in USA, Europe [37], Australia [38], and also some Asian countries [39,40]. ESBL producing Enterobacteriaceae isolates showed higher rates of resistance to all studied antimicrobials compared to the non-ESBL-producing isolates except for imipenem, where all tested isolates were imipenem-sensitive, that agrees with other Egyptian studies [30,33]. On the other context, a recent study from our region reported that, (31%) of K. pneumoniae isolated from hospital infections were resistant to imipenem [41]. Although MDR rate among ESBL producers in   [35], Iran [38], Qatar [40] and Japan [43]. blaTEM and blaSHV-producing strains were reported previously as hospital pathogens until the late 1990s [42], however blaTEM and blaSHV gene were highly frequent among our isolates (189, 60.7%) and (133, 42.8%) respectively, this may be caused by previous contact with health care workers. This higher frequency of blaTEM gene in our report and also in a recent report from our region may indicate that blaTEM gene may be endemic in our locality [30]. Co-carriage of multiple ESBL genes in the same isolate was detected previously in Egypt [29,30] and other countries; Burkina Faso [35], Qatar [40] and Iran [44], that concurs with our study, where 235 isolates (75.5%) harbored blaCTX-M in combination with blaTEM and/or blaSHV genes. AmpC β-lactamase production was identified phenotypically in 35 (7.9%) of the study isolates that was comparable with previous studies in Egypt [15,45] and neighboring countries [46,47]. However, another previous study in Egypt reported a higher rate (76.9%) [48].  [45,48]. Co-carriage of AmpC genes was found exclusively in K. pneumoniae isolates that agrees with previous reports from Egypt and North Africa [15,49]. Although FOX gene was commonly detected in previous Egyptian studies [15,48], it is not detected at all in the current study. ESBL genes were detected in 17/18 (94.4%) of AmpC genes-carrying isolates, that was also reported previously [50]. The spread of ESBL genes is related to different mobile genetic elements, such as plasmid, transposons, and integrons. The co-carriage of ESBL and other-resistant genes in the same transposable genetic elements explain the co-resistance of ESBL producers to variable antibiotics. Our study investigated the frequency of aac(6′)-Ib gene among ESBL-producing Enterobacteriaceae, that was high rate (53%), particularly among blaCTX-M-carrying strains (75.5%). The association between aac(6′)-Ib-cr gene and blaCTX-M genes was statistically significant (p-value < 0.01). This finding may explain why resistance to CIP, CN and AK was significantly higher in ESBL producers than in the non-ESBL-producers, that findings are compatible with several previous studies [39,51,52].

Conclusion
Our study detected high prevalence of ESBL-production among isolated from community-acquired UTIs in south Egypt, however the prevalence AmpC