Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum β-lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan

Background: This study aimed to characterize blaCTX-M, blaTEM, and blaSHV genes among extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae species in different regions of Sudan. Methods: In this cross-sectional study, different clinical samples (n = 985) were collected randomly from symptomatic patients from four geographical regions of Sudan and cultured on chromogenic media. Following bacterial identification, phenotypic screening of ESBLs was done according to CLSI guidelines using cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. The DNA was extracted by guanidine hydrochloride protocol, and then conventional PCR was used to detect blaCTX-M, blaTEM, and blaSHV genes. The presence of genes’ subtypes was characterized by DNA Sanger sequencing for selected samples. Results: Enterobacteriaceae represented 31% (305/985) of all isolates, 42 (128/305) of which were ESBLs producer, confirmed by phenotypic confirmatory test (75% [96/128] of them were positive for blaCTX-M genes, 61% [78/128] for blaTEM genes, and 38% [48/128] for blaSHV genes). Fourteen isolates (11%) were negative for all genes. Forty-eight percent (63/75) of Escherichia coli isolates were positive for blaCTX-M, while in Klebsiella pneumoniae, the dominant gene was blaTEM (82%) and had a low amount of blaSHV (59%). There was a significant association (P-value = 0.001 for all except for chloramphenicol, P = 0.014, and amikacin, P = 0.017) between resistance to third-generation cephalosporins and ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin. Forty-two percent (40/96) of CTX-M-positive isolates were in Gizera State, 33% (32.96) in Sinnar, 24% (23/96) in Khartoum, and 1% (1/96) in White Nile. Conclusion:We conclude that blaCTX-M genes are the most dominant genes in ESBLsproducing isolates and are more prevalent in big cities than in rural areas.


Introduction
Extended-spectrum beta-lactamases (ESBLs) are the types of enzymes that cause resistance to most beta-lactam ring containing antibiotics [1]. Enterobacteriaceae sp. can resist a wide range of antibiotics, including cephalosporin and carbapenems, used as last-line antibiotics [2]. Infections caused by ESBLs-producing Enterobacteriaceae are increasingly being reported worldwide, causing high mortality rates, prolonged hospital stay, and rising medical costs [3]. Escherichia coli and Klebsiella pneumoniae species are considered among species associated with the high spread of ESBLs genes globally, especially the bla CTX-M genes, which have become more common in the last 20 years.
Recently, a dramatic increase has been reported in the frequency of bla CTX-M types β-lactamases-producing bacteria, which replaced the predominant types in the past, such as bla TEM and bla SHV [4]. bla CTX-M carrier, E. coli, can disseminate these genes in the community and hospitals from intestinal flora and cause infection [5].
Sudan is one of the many developing countries suffering from irrational use of antibiotics, where 63% of prescriptions contain antibiotics, and various forms of irrational cephalosporins usage are noticed [6,7]. In Sudan, there is no regulation or system to govern antimicrobial use in humans or animals [8]. Resistance to cephalosporins and production of ESBLs genes in hospitals and environment have been reported in previous studies [9][10][11]. For the first time, this study aimed to detect bla CTX-M , bla SHV , and bla TEM genes and their subtypes among ESBLs-producing Enterobacteriaceae in different regions of Sudan.

Phenotypic detection of ESBLs
Phenotypic screening of ESBLs was done according to CLSI guidelines, the following discs were used: cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. Phenotypically, ESBLs-positive isolates showed an increase of ≥5 mm in the zone around discs with clavulanic acid discs compared to the area around the disc without clavulanic acid [13].

Identification of extended-spectrum -lactamase genes
All positive isolates with phenotypic confirmatory tests were subjected to molecular screening to detect β-lactamases genes using a conventional PCR machine. DNA isolation was done by the guanidine hydrochloride method, according to Sabeel et al. [14]. PCR was carried out using primer sequences presented in Table 1 (Metabion, Germany) for bla CTX-M , bla TEM , and bla SHV genes [15,16]. A reaction volume of 25 μl containing 5 µl Master Mix (iNtRON Biotechnology, Seongnam, Korea), 2 µl DNA, 0.6 μl of each primer, and 16.8 µl DW was used. The PCR steps were firstly subjected to 94°C for 5 min, then 30 cycles (94°C for 45 sec, 57°C for 45 sec, 72°C for 60 sec), and final elongation at 72°C for 5 min. PCR products were run at 2% agarose gel for bands detection by UV Transilluminator. Control positive (obtained from previously sequenced bla CTX-M , bla TEM , and bla SHV genes) and control negative (containing DW, primers, and Master Mix) were used.
Sanger sequencing was achieved for both directions of DNA products by Macrogen Company (Seoul, Korea). DNA sequencing was performed for 25 bla CTX-M (7 from Sinnar, 5 from Khartoum, 1 from White Nile, and 12 from Gizera), 3 bla SHV , and 4 bla TEM genes.

Statistical analysis
Data were analyzed by the statistical package for social science (SPSS) version 16, using the Chi-square test. A P-value < 0.05 was considered significant.

Results
While 68% ( (Table 3). There was a statistically significant (P-value < 0.05) association between ESBLs genes and the different regions of Sudan.  Table 4). Twenty-four isolates possessed only bla CTX-M genes, seven only bla TEM genes, and one only bla SHV gene. Twenty-six isolates possessed the three genes together, seven harbored bla TEM and bla SHV genes, twentynine possessed both bla CTX-M and bla TEM genes, and ten harbored bla CTX-M and bla SHV genes. Moreover, 14 isolates (11%) gave negative results for the three genes.   The association between antibiotic resistance and ESBLs-producing and non-ESBLs-producing bacteria.

Discussion
Several studies exhibited that the prevalence of ESBL-producing bacteria is a serious problem of global public health, and their distribution can be varied according to geographic region, country, and studied institution [17,18].  In this study, we report the increasing rates of ESBLs-producing Enterobacteriaceae compared to other previous study conducted in Khartoum State by Mekki et al. [19], we recorded ESBLs production among Klebsiella sp. and E. coli isolated as 53% in 2010. Moreover, Ahmed et al. [20] had recorded ESBLs production among Enterobacteriaceae sp. as 59.6% in 2013. This finding in the Sinnar State is higher than that previously reported in 2012 by Hamedelnil and Eltayeb [21], who reported that 36% of 133 isolates were ESBLs producers.
In the current study, we report that resistance to cefotaxime (45%) is higher than that to ceftazidime (39.3%) and cefepime (13.4%). We also observed a high ceftazidimeresistant rate (39.3%) within CTX-M-positive isolates, which may be because we report high frequencies of bla CTX-M genes in our isolates. Previous reports indicated several types of bla CTX-M genes exhibiting an increased enzymatic activity against ceftazidime [22,23]. A high resistance rate was observed in this study to ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin within ESBLs-producing isolates. There was statistically significant (P-value < 0.05) association between resistance to thirdgeneration cephalosporins and these antibiotics (P-value = 0.001, 0.001, 0.001, 0.014, and 0.017, respectively). The possible cause for this phenomenon may be that ESBLs are encoded on mobile plasmids, facilitating its transmission from one organism to another [24]. and ESBLs phenomenon may arise from another mechanism of resistance.
In the present study, we observed that the cephalosporin-resistance rate and produc- Also, antibiotic consumption is higher in cities than in rural areas due to easy access to hospitals and pharmacies, and this is observed in the result of Abu Rugba village, where there is no pharmacy or hospital. Unfortunately, in Sudan, cephalosporins and other antibiotics are sold as over-the-counter medication, explaining the overuse of antibiotics [6,7].

Limitations
Limited resources prevented us from sequencing all amplified genes, and we selected some samples from different regions to be used as control strains to give us a general idea about the common gene subtypes.

Conclusions
This study has shown a high prevalence of ESBLs-producing bacteria in different regions of Sudan, especially in big cities than in rural areas. bla CTX-M genes are the most dominant genes in ESBLs-producing isolates. This alarming situation of explosive spreading of ESBLs genes, especially blaCTX − -producing isolates, highlights the need for their epidemiological monitoring. Integrated and regular management of antibiotic consumption needs to be monitored in our society to limit their spread.