Distribution of extended-spectrum beta-lactamase TEM and CTX-M resistance genes among Proteus species Isolated in Sudan
Distribución de genes de resistencia de betalactamasas de espectro extendido TEM y CTX-M entre especies de Proteus aisladas en Sudán
Hassan A. Musa,1* Mayada A.M. Osman,1 Yasir H. Abdelaziz,2 Salma Mohamed,3 Mohammed Ibrahim-Saeed,3
1 Microbiology Dept. Faculty of Medicine. The National Ribat University, Sudan.
2 Microbiology Dept. Faculty of Medicine. Razi University, Sudan.
3 Microbiology Dept. Faculty of Medical Laboratory Science. The National Ribat University, Sudan.
* Professor. Diploma in Bacteriology. Department of Microbiology. The National Ribat University, Sudan.
Proteus species are found in the human intestinal tract as part of normal flora. Proteus species are also found in multiple environmental habitats, including long-term care facilities and hospitals, and can cause both community and nosocomial infections. For a long time Proteus was known to be susceptible to beta-lactam antibiotics but nowadays they become resistant. The aim of this study was to detect the Extended-spectrum beta-lactamase (ESBL) TEM and CTX-M genes in 90 Proteus species isolated from urine and wound swabs, obtained from different hospitals in Khartoum state, Sudan, from January to August 2018. Antimicrobial sensitivity was carried out using the following set of antibiotics: amoxiclav, ceftazidime, gentamicin, meropenem, cefotaxime, ciprofloxacin, amoxicillin, ceftriaxone and cotrimoxazole. ESBL producing strains were detected by double disc diffusion synergy test and the resistance genes TEM and CTX-M were detected by Polymerase Chain Reaction (PCR). Antibiotic resistance was found: amoxicillin 40%, ceftazidime 25.6%, ceftriaxone 23.3%, gentamicin 22.2%, cotrimoxazole 21.1%, and cefotaxime 18.9%. Most of the isolates were sensitive to meropenem 92.2% and ciprofloxacin 86.7%. In double-disk diffusion synergy test, 20 isolates (22.2%) were found to be positive for ESBL. The PCR demonstrated that TEM gene was present in 18 isolates (90%). It was present alone in 11 isolates (55%) and in combination with CTX-M gene in seven isolates (35%). The percentage of ESBL producing strains of Proteus was 23.5%. This percentage is a bit lower than in previous studies in Sudan. In conclusion; it seems that the CTX-M gene is emerging among Proteus species in Sudan.
Keywords: Sudan; proteus; beta-lactamase; genes; nosocomial infections.
Las especies de Proteus se encuentran en el tracto intestinal humano y forman parte de su flora normal. También se localizan en el medio ambiente y otros hábitats, incluyendo hospitales y diversas instituciones de salud, provocando tanto infecciones en la comunidad como nosocomiales. Durante mucho tiempo, las especies de Proteus fueron susceptibles a los antibióticos betalactámicos, pero actualmente se han tornado resistentes. El propósito de este estudio fue detectar genes de resistencia betalactamasas de espectro extendido (BLEE) TEM y CTX-M, en 90 especies de Proteus aisladas en orina y heridas, provenientes de diversos hospitales del estado de Jartum, Sudán, entre enero y agosto de 2018. La sensibilidad antimicrobiana se determinó con el siguiente juego de antibióticos: amoxiclav, ceftazidima, gentamicina, meropenem, cefotaxima, ciprofloxacina, amoxicilina, ceftriaxona y cotrimoxasol. Las cepas productoras de BLEE se detectaron mediante la técnica de sinergia de doble disco, y los genes de resistencia TEM y CTX-M mediante Reacción en Cadena de la Polimerasa (PCR). Se encontró resistencia antibiótica: amoxicilina 40%, ceftazidima 25,6%, ceftriaxona 23,3%, gentamicina 22,2%, cotrimoxasol 21,1% y cefotaxima 18,9%. La mayor parte de los aislamientos fueron sensibles a meropenem (92,2%) y ciprofloxacina (86,7%). Con la técnica de sinergia de doble disco se detectó positividad a BLEE en 20 aislamientos (22,2%). Mediante PCR se demostró que el gen que codifica TEM estaba presente en 18 aislamientos (90%); de forma aislada en 11 aislamientos (55%) y combinado con el gen CTX-M en los otros siete (35%). El porcentaje de cepas de Proteus productoras de BLEE fue de 23,5%. Este valor es ligeramente inferior que los detectados en estudios previos en Sudán. En conclusión, hay evidencias de que el gen CTX-M está emergiendo entre las especies de Proteus en Sudán.
Palabras clave: Sudan; Proteus; betalactamasa; genes; infecciones nosocomiales.
Infectious diseases continue to be a major cause of morbidity and mortality.(1)(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.
1. Cloherty JP. Maternal conditions that affect the fetus: Diabetes mellitus. In Cloherty JP, Stark. AR, editors. Manual of neonatal care 4th edition, Philadelphia: Lippincott-Raven Publishers; 1998. p.15-9.
2. Calil R, Marba ST, von Nowakonski A, Tresoldi AT. Reduction in colonization and nosocomial infection by multiresistant bacteria in a neonatal unit after institution of educational measures and restriction in the use of cephalosporins. Am J Infect Control. 2001;29(3):133-8.
3. Khan MK, Thukral SS, Gaind R. Evaluation of a modified double-disc synergy test for detection of extended spectrum ß-lactamases in AMPC ß-lactamase-producing Proteus mirabilis. Indian J Med Microbiol. 2008;26(1)58-61.
4. Al-Muharrmi Z, Rafay A, Balkhair A, Jabri AA. Antibiotic combination as empirical therapy for extended spectrum Beta-lactamase. Oman Med J. 2008;23(2):78-81.
5. Nathisuwan S, Burgess DS, Lewis JS. Extended-spectrum ß-lactamases: epidemiology, detection, and treatment. Pharmacotherapy: Pharmacotherapy 2001;21(8):920-8.
6. Bali EB, Acik L, Sultan N. Phenotypic and molecular characterization of SHV, TEM, CTX-M and extended-spectrum beta-lactamase produced by Escherichia coli, Acinobacter baumannii and Klebsiella isolates in a Turkish hospital. Afr J Microbiol Res. 2010;4(8):650-4.
7. Seyedjavadi SS, Goudarzi M, Sabzehali F. Relation between blaTEM, blaSHV and blaCTX-M genes and acute urinary tract infections. Journal of Acute Disease. 2016;5(1):71-6.
8. Coyle MB. Manual of antimicrobial susceptibility testing. Washington DC: American Society for Microbiology; 2005.
9. Mordi RM, Momoh MI. Incidence of Proteus species in wound infections and their sensitivity pattern in the University of Benin Teaching Hospital. Afr J Biotechnol. 2009;8(5):725-30.
10. Jones RN, Baquero F, Privitera G, Inoue M, Wiedemann B. Inducible ß-lactamase-mediated resistance to third-generation cephalosporins. Clin Microbiol Infect. 1997;3(Suppl 1):S7-S20.
11. Gualerzi CO, Brandi L, Fabbretti A, Pon CL, editors. Antibiotics: targets, mechanisms and resistance. Weinheim, Germany: Wiley-VCH; 2014.
12. Roe BA, Crabtree JS, Khan AS. DNA isolation and sequencing. New York: Wiley-Blackwell; 1996.
13. Ahmed OI, El-Hady SA, Ahmed TM, Ahmed IZ. Detection of bla SHV and bla CTX-M genes in ESBL producing Klebsiella pneumoniae isolated from Egyptian patients with suspected nosocomial infections. Egypt J Med Hum Genet. 2013;14(3):277-83.
14. Hassan RM, Hammad MN. Prevalence and Antimicrobial Susceptibility Pattern of Extended Spectrum ß-Lactamases Producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Khartoum Sudan. Am J Res Commun. 2016;4(8):60-6.
15. Ahmed OB, Omar AO, Asghar AH, El Hassan MM. Increasing prevalence of ESBL-producing Enterobacteriaceae in Sudan community patients with UTIs. Egypt Acad J Biolog Sci. 2013;5(1):17-24.
16. Bauernfeind A, Holley M, Jungwirth R, Mangold P, Röhnisch T, Schweighart S, et al. A new plasmidic cefotaximase from patients infected with Salmonella typhimurium. Infection. 1992;20(3):158-63.
17. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty Informational Supplement. CLSI document M100-S20. Wayne, PA: CLSI; 2010
18. Fazeli H, Dolatabadi RK, Taraghian A, Isfahani BN, Nasr Nasr B, Moghim SH, et al. Carbapenem Resistance Pattern of Multiple Drug-Resistantand Extended-Spectrum Beta-Lactamase-Positive Klebsiella pneumonia in Isfahan. Int J Enteric Pathog. 2014;2(4):e21495. Doi:10.17795/ijep21495
19. Miriagou V, Cornaglia G, Edelstein M, Galani I, Giske CG, Gniadkowski M, et al. Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues. Clin Microbiol Infect. 2010;16(2):112-22.
20. Endimiani A, Luzzaro F, Brigante G, Perilli M, Lombardi G, Amicosante G, et al. Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum ß-lactamases. Antimicrob Agents Chemother. 2005;49(7):2598-605.
21. Hindi AK. Phenotyping and Molecular Characterization of Proteus vulgaris Isolated from Patients with Urinary Tract Infections [dissertation]. Babylon, Irak: College of Science, University of Babylon; 2014.
22. Caubey M, Suchitra M. Occurrence of TEM, SHV and CTX-M ß Lactamases in Clinical Isolates of Proteus Species in a Tertiary Care Center. Infect Disord Drug Targets. 2018;18(1):68-71.
Conflicts of interest
The authors declare there are no conflicts of interest.
Submitted: March 15, 2019 Approved: June 3, 2019