Introduction
Infectious diseases continue to be a major cause of morbidity and mortality.1 Multidrug-resistant members of the family of Enterobacteriaceae are responsible for such infections.2 Patients with infections due to extended-spectrum beta-lactamase (ESBL) producing organisms are likely to have a poor outcome when compared to those infected with non-producing organisms.3 ESBL are enzymes produced by many gram-negative bacteria. They have the ability to inactivate the third generation cephalosporin, penicillin, and the monobactam antibiotics. However, they are inhibited by clavulanic acid.4,5
Several investigations have reported a different prevalence of ESBLs ranging from 6% to 88% in various health care setting especially among members of Enterobacteriaceae. Although TEM, SHV genes were the most common ESBL producing genes.6,7 Most of the gram-positive bacteria produce their beta-lactamases in the surrounding, thus inactivating beta-lactam antibiotics externally. And by contrast, the beta-lactamases of the gram-negative bacteria remain inside the cells inactivating the drug in the periplasmic space.8
Proteus species are members of the family Enterobacteriaceae, present as normal flora of the human intestine and in various environmental habitats including hospitals. They can cause both community and nosocomial acquired infections.9 For a long time, Proteus was known to be susceptible to beta-lactam antibiotics. Nowadays they are becoming resistant due to the spread of extended-spectrum beta-lactamase.10 The aim of this study was to detect the beta-lactamase TEM and CTX-M genes in clinical isolates of Proteus species from different hospitals in Khartoum state, Sudan.
Materials and Methods
This prospective hospital-based cross-sectional study was carried out from January to August 2018. Ethical considerations of the research objectives were approved by the National Ribat University ethical committee. Voluntary informed consent form was signed by each participant after explaining the objectives of the study. Ninety Proteus strains were isolated from urine and wound swabs, the isolation and identification of pure culture were achieved based on colonial morphology, gram stain and set of biochemical tests. Isolates were selected on the following profile of identification: gram negative bacilli, non-lactose fermenter and urease positive.
Antimicrobial sensitivity was carried out according to the CLSI method using the following set of antibiotics: amoxiclav (AMC), ceftazidime (CAZ), gentamicin (GN), meropenem (MEM), cefotaxime (CFM), ciprofloxacin (CIP), amoxicillin (AX), ceftriaxone (CRO) and cotrimoxazole (STX).11
ESBL producers were detected by a double disc diffusion synergy test.11 The TEM and CTX-M genes were detected by the DNA guanidine chloride extraction.12 The Polymerase Chain Reaction (PCR) was done using primers for the TEM and CTX-M genes (Table 1). The reaction mix and procedure protocol and optimization were applied according to product instruction sheet (Promega Corporation, USA). The results of antibiotics susceptibility were calculated based on the percentage of the number of isolates that show sensitivity or resistance toward each antibiotic divided by the total number of isolates (90).
Results and Discussion
The results of antibiotic sensitivity showed that most of the isolated strains were sensitive to meropenem: 83 (92.2%) and ciprofloxacin: 78 (86.7%) (Fig. 1). While the highest antibiotic resistance were obtained with amoxicillin: 36 (40%), ceftazidime: 23 (25.6%), ceftriaxone: 21 (23.3%), gentamicin: 20 (22.2%), cotrimoxazole: 19 (21.1%), and cefotaxime: 17 (18.9%) (Fig. 2).
Twenty isolates (22.2%) were found to be positive for ESBL by double-disk diffusion synergy test, then ESBL resistant genes were detected by PCR using selected primers. TEM and CTX-M genes were present in 18 isolates (90%). TEM was present alone in 11 isolates (55%) and CTX-M gene was present in seven isolates (35%) (Fig. 3).
None of the CTX-M genes were detected separately. While two isolates out of the 20 ESBL producers were negative for both TEM and CTXM genes (10%). The common pattern of resistance associated with both the TEM and CTX-M gene carriers was a combination of ceftazidime, ceftriaxone and amoxicillin (83.3%).
The presence of ESBL producing bacteria is striking rapid worldwide, hence the increase of resistance to antibiotics and the emergence of multidrug-resistant ESPL producers are becoming a public health problem, causing clinical failure of empirical antibiotic treatment.13 Consequently, a continuous monitoring system and effective control measures are absolutely required.
The percentage of ESBL Proteus strains in this study was 22.2%. This percentage is a bit lower than in previous studies in Sudan, where the detected percentage was 29.6% in 2016 and 33.3% in 2013.14,15 It was even lower in comparison with a study in Turkey, where the percentage of ESPL producers were 48.5%.6
Plasmids with Multidrug-resistant genes are common among the family of Enterobacteriaceae. Historically, Proteus species were known to be free of the beta-lactamase genes.16 However, Proteus, as a member of the family Enterobacteriaceae, can acquire the plasmids from other members of the family. For a long time, Proteus species are known to carry the TEM beta-lactamase gene only.17 Nowadays, the currently spreading beta-lactamase is the CTX-M gene. Generally, the CTX-M gene is replacing the TEM gene and the SHV genes.
In this study, the TEM gene was detected alone in 55% of the ESBL producers and in 35% in combination with the CTX-M gene. It seems that the CTX-M gene is emerging among Proteus species. In various studies conducted in India, Proteus species were carrying the TEM gene only.18,19 In Italy, 44% of the ESBL producing Proteus were also carrying the TEM gene only.20 While in Iraq, all ESBL producing Proteus had this gene.21 In recent studies performed in India, 35.3% of the ESBL producing Proteus were carrying CTX-M gene alone; the 1.8% of strains the TEM gene and the 52.9% both genes together.22
The CTX-M gene appears to emerge in combination with TEM gene at the beginning and then replacing the others as the dominant gene is spreading. It seems that the selective pressure by the misuse of antibiotics has created a favorable environment for the spread of ESBLs among Enterobacteriaceae.