|Year : 2018 | Volume
| Issue : 1 | Page : 19-23
Molecular Characterization and Antibiotic Susceptibility Pattern of Acinetobacter Baumannii Isolated in Intensive Care Unit Patients in Al-Hassa, Kingdom of Saudi Arabia
Mohammed Sami Alhaddad1, Afnan Khalifah AlBarjas1, Lolowah Ebraheem Alhammar1, Abdullatif Sami Al Rashed1, Lorina Ineta Badger-Emeka2
1 College of Medicine, King Faisal University, Al-Hassa, Kingdom of Saudi Arabia
2 Department of Biomedical Sciences, Division of Microbiology, College of Medicine, King Faisal University, Al-Hassa, Kingdom of Saudi Arabia
|Date of Submission||19-Mar-2017|
|Date of Acceptance||16-Oct-2017|
|Date of Web Publication||13-Feb-2018|
Dr. Lorina Ineta Badger-Emeka
Department of Biomedical Sciences, Division of Microbiology, College of Medicine, King Faisal University, PO Box 400, Al-Ahsa 31982
Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Acinetobacter baumannii, is an emerging nosocomial multidrug resistance pathogen with the rapid spread of clones being reported in health-care settings and hospitals worldwide. Carbapenem resistance in this bacterium has been attributed to D OXA β-lactamases with OXA-51-like β-lactamase, being present in all A. baumannii isolate. The present study looks into the antibiotics susceptibility and molecular characterization of clinical A. baumannii isolates from Intensive Care Unit (ICU) samples in Al-Hofuf, South-eastern region of Saudi Arabia. Materials and Methods: Eleven strains of ICU A. baumanni i isolates were used for the investigation. Bacteria isolation was by basic microbiological techniques. Organisms identification and antibiogram susceptibility testing was by the BioMerieux VITEK 2 compact automated system (BioMerieux, Marcy I'Etoile France), according to the manufacturers guidelines. Confirmation of A. baumannii was by the presence of the OX-51 gene, also, carbapenemase encoding resistant genesblaOXA-23, blaOXA-40, and blaOXA-51, were analyzed using multiplex PCR. The Student's t test was used to analyze the obtained data for between group comparisons with statistically significance level set at P < 0.05. Results: Eight of the isolates were confirmed to be A. baumannii. Five of which were resistant to the carbapenems against which they had been tested. One isolate was resistant to tigecycline, whereas three tested intermediate to the drug. OXA-23 was detected in isolates 1, 4, 5, 6, and 7. Conclusion: It can, therefore, be concluded that the probable predominate carbapenems resistant genes in ICU isolates from the present investigation, are those associated with OXA-23.
Keywords: Acinetobacter baumannii, Intensive Care Unit, multidrug resistance, OXA-23, OXA-51, extensive drug resistance
|How to cite this article:|
Alhaddad MS, AlBarjas AK, Alhammar LE, Al Rashed AS, Badger-Emeka LI. Molecular Characterization and Antibiotic Susceptibility Pattern of Acinetobacter Baumannii Isolated in Intensive Care Unit Patients in Al-Hassa, Kingdom of Saudi Arabia. Int J App Basic Med Res 2018;8:19-23
|How to cite this URL:|
Alhaddad MS, AlBarjas AK, Alhammar LE, Al Rashed AS, Badger-Emeka LI. Molecular Characterization and Antibiotic Susceptibility Pattern of Acinetobacter Baumannii Isolated in Intensive Care Unit Patients in Al-Hassa, Kingdom of Saudi Arabia. Int J App Basic Med Res [serial online] 2018 [cited 2018 Apr 22];8:19-23. Available from: http://www.ijabmr.org/text.asp?2018/8/1/19/225449
| Introduction|| |
The increase in difficult to treat multi-drug resistant bacteria has created an unprecedented problem in the 21st century. Acinetobacter baumannii has been listed among the most difficult antimicrobial resistant Gram-negative bacilli.,A.baumannii an opportunistic pathogen has evolved to be an important pathogen associated with nosocomial infections worldwide and is known to have developed strains that are multidrug resistance (MDR), extensive drug resistance (XDR) and Pandrug resistance. Researchers ,,,, have carried out extensive investigations into various aspects of this bacteria supper bug in which they had looked into the various genes responsible for carbapenemase resistance. A.baumannii is postulated to have emerged as a nosocomial pathogen with the rapid spread of clones being reported in hospitals and health-care settings ,, Outbreaks of MDR A.baumannii in critical care units have also been reported in various regions of the world., Furthermore, colonization with this bacterium in immunocompromised patients have been reported by researchers , with diabetes being shown to be a significant risk factor for A.baumannii nosocomial infections.,, Other risk factors which have been associated with patient colonization by A.baumannii are exposure to ICUs as well as prolonged hospital stays. Patients at risk could be through exposure to mechanical ventilation, recent surgery, invasive procedures , as well as items used in the care of patients.,
The carbapenems had been the drug of choice in treatment due to their effective antibacterial activity as well as having low toxicity. However, A.baumannii has emerged resistant to this drug of choice resulting in a major global health concern., Resistance to the carbapenem manifested in plasmid-encoded β-lactamase (OXA-23, OXA-40, and OXA-58) enzymes and the chromosomally encoded OXA-51. The clones harbored by different A.baumannii isolates have been shown to differ. Differences have been indicated between the isolates diabetic and nondiabetic patients  with no specifics indicating if there are clones associated with diabetes. The present work looks into A.baumannii isolates from Intensive Care Unit (ICU) samples with a view of providing an insight in the prevalent OXA β-lactamase associated with ICU isolates. This would help provide information on the genes associated with ICU isolates as there is the need for constant surveillance in the microbial susceptibility of bacteria isolates in this era of evolving bacteria superbugs.
| Materials and Methods|| |
Sample collection and processing
Clinical isolates on A.baumannii obtained from ICU samples were used for the study. Samples had been isolated from sputum, throat, abdomen, catheter and endotracheal aspirates and plated out on MacConkey agar. A.baumannii isolation and identification was carried out in hospitals using basic microbiological and biochemical techniques.
Isolation was through basic microbial techniques while confirmation of isolates was carried out using BioMerieux VITEK 2 compact automated system (BioMerieux, Marcy I'Etoile France), according to the manufacturer's guidelines. For further confirmation, PCR was used to detect the intrinsic blaOXA-51- gene.
Antimicrobial susceptibility testing
Pure samples grown overnight on MacConkey agar were used for the antibiogram testing. The antibiotic susceptibility testing was carried out using the VITEK 2 compact automated system according to manufacturer's guidelines. Isolates were tested against the GN cards with the following antibiotics: cefazolin, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, tigecycline, and sulfamethoxazole/trimethoprim. Only isolates which showed resistance to imipenem and meropenem were used for the investigations. The minimum inhibitory concentration values were determined using the ETest strips (AB Biodisks) following the manufacturer's guidelines. The test experiments were repeated in triplicates.
Molecular analysis for carbapenem-resistant genes
The presence of carbapenemase encoding resistant gene (blaOXA-23, blaOXA-40, blaOXA-51) among the isolates were detected using multiplex PCRgenes using primers pairs.,,, The primers used for the amplification and sequencing are shown in [Table 1].
The PCR protocol is as described by Marco et al. Samples were initially denatured for 5 min at 94°C, followed by amplification cycles and elongation steps as described by Marco et al. Resulting PCR products were visualized after agarose gel electrophoresis, staining with ethidium bromide.
Excel software, 2013 version was used to analyze the obtained data, with student's T-test used for comparison between groups. Statistically, significance level was set at P < 0.05.
| Results|| |
A total of eight nonrepetitive confirmed isolates of A.baumannii collected from ICUs in the study region were used for the investigation. The isolate source and the minimum inhibitory concentration (MIC) values are shown in [Table 2]. The result on the susceptibility of these isolates against tested antibiotics is presented in [Table 3]. The isolates are seen to exhibit varying degrees of resistance to the different antibiotics with 56% of them being resistant to imipenem and meropenem. However, all (100%) the isolates were sensitive to colistin, One (12.5%) isolate was resistant to tigecycline, whereas 33.33% of the isolates were intermediate to tigecycline. Based on their resistance against the tested antibiotic groupings, 67% of the isolates are MDR. [Figure 1] shows the results of the resistance of these isolates to the antibiotic categories. Highest resistance was against the cephalosporins (78%) followed by fluoroquinolones with a 67% resistance by the isolates. Fifty-six percent of the A.baumannii were each resistant to the carbapenems and penicillin, whereas 44% of them were each resistant to the aminoglycosides and trimethoprim/sulfamethoxazole.
|Table 2: Source of isolation and the minimum inhibitory concentration vales against tested antibiotics|
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|Table 3: Antibiogram of multidrug resistance clinical Acinetobacter baumannii isolates from Intensive Care Units|
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|Figure 1: Percentage resistance of Acinetobacter baumannii to anti-microbial categories. STX = Sulphamethoxazole/trimethoprim|
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A comparison between the number of antibiotics that the isolates were resistant and sensitive to is shown in [Table 4]. For isolate 1, there was about a 50% resistance against the tested antibiotics. The difference between the number of antibiotics that the isolate was resistant or sensitive to is not statistically significant. However, for isolates 3, 4, 5, and 7, there was a 75% to 80% resistance against the tested antimicrobials, inclusive of the carbapenems. The results are statistically significant (P < 0.05). Isolates 8 and 9 were more susceptible to most of the tested drugs and the difference against the drugs to which they were resistant to was not statistically significant as shown in [Table 4].
|Table 4: Comparison between resistance and sensitivity against the tested antibiotics by Intensive Care Unit isolates of Acinetobacter baumannii|
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On the other hand, for the comparison between the antibiotics to which isolates were resistant or intermediate to, results showed that there is a statistically significant difference (P < 0.05) with the exception of isolate 1 that recorded no statistical difference [Table 5]. Also, the results on the prevalent genes seen to be associated with the ICU A. baumannii isolates is shown in [Figure 2]. The OXA-23 was present in 56% of the isolates while OXA 40 was not encountered in any of the isolates.
|Table 5: Relationship between intermediate and resistance to tested antibiotics among Intensive Care Unit Acinetobacter baumannii tes|
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|Figure 2: Characterisation of carbapenemase genes. The OXA-23 enzyme was detected in 5 (56%) of the isolates. All the isolates were negative to OXA-40|
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| Discussion|| |
The health-care problem caused by MDR A.baumannii is further highlighted in the results of the present investigation. The isolation of MDR A.baumannii from ICU samples as seen in the present study is not unexpected as such isolations had been reported earlier by researchers.,,, Furthermore, the evolvement of both MDR and XDR A.baumannii has been reported by Kempf and Rolain in 2012.
The results from the present study also showed majority of the isolates to be MDR, whereas >50% of them showed XDR against the tested antibiotic groups. The exhibition of high levels of MDR and XDR by A.baumannii isolates has been reported in different regions of the world. Nageeb et al. reported a 100% MDR and 80% XDR among A.baumannii isolates obtained from ICU in Egypt, whereas in Pakistan, Evans et al., encountered 82.4% MDR and 65% XDR among the isolates in their study. Both findings differ from those of the present investigation. However, Dent et al., reported a 72% and 58% MDR and XDR, respectively, among A.baumannii in their study Nashville General Hospital and this finding is similar to those of the present study. While being certain that all the A.baumannii ICU isolates in the present study showed both MDR and XDR against the tested antibiotic groups, the observed difference between researches could be attributed to a number of contributing factors one of which is geographical regional isolate differences, as well as the total number of isolates being used for the investigations by the different researchers as had been indicated by Nageeb et al.
Furthermore, the resistance by more than half of the isolates to the carbapenems and other β-lactams as seen in the present investigation indicates that carbapenem resistance remains a problem. Al-Sultan et al. had reported that carbapenem resistance was very high among A.baumannii hospitals isolates in Saudi Arabia. The OXA-51- and OXA-23-like genes were the encountered genes responsible for carbapenem resistance in the A.baumannii isolates in the present study as shown in [Figure 2]. Yaowen et al. observed OXA-23 gene to be the major carbapenemase responsible for resistance in their isolates from a teaching hospital. The findings are also consistent with those of Merino et al. and Zowawi et al. Observed in these carbapenem-resistant ICU isolates was the carriage of both the OXA-23 and OXA-51 genes. Similar findings had been reported by Al-Sultan et al., in 2013 on A.baumannii isolates from diabetic patients. The presence of both genes was however detected in one isolate which was susceptible to both imipenem and meropenem. This isolate was also susceptible to other β-lactams, fluoroquinolone and intermediate to aminoglycoside. Similar to this finding is that of Yaowen et al., that reported an A.baumannii isolate which had been susceptible to both imipenem and meropenem but was found to be carrying the blaOAX-72 gene with no other resistant gene. This isolate was reported as susceptible to other β-lactams, aminoglycosides, and fluoroquinolones among other antibiotics. The authors concluded however that there was a need for more study to find out why the presence of OXA-72 did not produce β-lactam resistance. Furthermore, El-Shazy et al., in 2015 reported that not all the A.baumannii isolates in their study harboring the ISAbla1 gene were resistant to the carbapenems. Evans et al. found the OXA-51-like enzyme expressed in a sensitive A.baumannii isolates and explained that the enzyme demonstrated the capability to dramatically increase the MIC to carbapenem for an isolate. They also indicated that the OXA-51-like enzyme demonstrated only a weak hydrolytic activity against carbapenemase. These might therefore explain the reason for the results recorded with one of the isolate in the present study. However, there is need for further study on the nonconferring of resistance in the presence of OXA-like gene as earlier suggested.
| Conclusion|| |
From the results of the present investigation, it can be concluded that the predominant carbapenems resistant in the ICU A. baumannii isolates in the present investigation, are those of the OXA-23. There is however need for more investigation for both monitoring and further updating.
The authors would like to thank Dr. Abdulrahman Al-Sultan for his expertise advice, Mr. Hani Al-Rasasi and Ms. Hajer, for their technical assistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Maragakis LL, Perl TM. Acinetobacter baumannii
: Epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis 2008;46:1254-63.
Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al.
Bad bugs, no drugs: No ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009;48:1-2.
Christopher AF, Hora S, Ali Z. Investigation of plasmid profile, antibiotic susceptibility pattern multiple antibiotic resistance index calculation of Escherichia coli isolates obtained from different human clinical specimens at tertiary care hospital in Bareilly - India. Ann Trop Med Public Health 2013;6:285-9. [Full text]
Senok A, Garaween G, Raji A, Khubnani H, Kim Sing G, Shibl A, et al.
Genetic relatedness of clinical and environmental Acinetobacter baumanii
isolates from an intensive care unit outbreak. J Infect Dev Ctries 2015;9:665-9.
Ushizawa H, Yahata Y, Endo T, Iwashima T, Misawa M, Sonobe M, et al.
A epidemiological investigation of a nosocomial outbreak of multidrug-resistant Acinetobacter baumannii
in a critical care center in Japan, 2011-2012. Jpn J Infect Dis 2016;69:143-8.
Alsultan AA, Evans BA, Elsayed EA, Al-Thawadi SI, Al-Taher AY, Amyes SG, et al.
High frequency of carbapenem-resistant Acinetobacter baumannii
in patients with diabetes mellitus in Saudi Arabia. J Med Microbiol 2013;62:885-8.
El-Ageery SM, Abo-Shadi MA, Alghaithy AA, Ahmad MA, Alsharif NH, Alharbi SA, et al.
Epidemiological investigation of nosocomial infection with multidrug-resistant Acinetobacter baumannii
. Eur Rev Med Pharmacol Sci 2012;16:1834-9.
Qi C, Malczynski M, Parker M, Scheetz MH. Characterization of genetic diversity of carbapenem-resistant Acinetobacter baumannii
clinical strains collected from 2004 to 2007. J Clin Microbiol 2008;46:1106-9.
Jean SS, Hsueh PR, Lee WS, Chang HT, Chou MY, Chen IS, et al.
Nationwide surveillance of antimicrobial resistance among non-fermentative Gram-negative bacteria in Intensive Care Units in Taiwan: SMART programme data 2005. Int J Antimicrob Agents 2009;33:266-71.
Chen CM, Liu PY, Ke SC, Wu HJ, Wu LT. Investigation of carbapenem-resistant Acinetobacter Baumannii
isolates in a district hospital in Taiwan. Diagn Microbiol Infect Dis 2009;63:394-7.
Wilks M, Wilson A, Warwick S, Price E, Kennedy D, Ely A, et al.
Control of an outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus
colonization and infection in an intensive care unit (ICU) without closing the ICU or placing patients in isolation. Infect Control Hosp Epidemiol 2006;27:654-8.
Navon-Venezia S, Leavitt A, Carmeli Y. High tigecycline resistance in multidrug-resistant Acinetobacter baumannii
. J Antimicrob Chemother 2007;59:772-4.
Prata-Rocha ML, Gontijo-Filho PP, Melo GB. Factors influencing survival in patients with multidrug-resistant Acinetobacter baumannii
infection. Braz J Infect Dis 2012;16:237-41.
Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek CJ, et al.
National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: Systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2·7 million participants. Lancet 2011;378:31-40.
Fournier PE, Richet H. The epidemiology and control of Acinetobacter baumannii
in health care facilities. Clin Infect Dis 2006;42:692-9.
Playford EG, Craig JC, Iredell JR. Carbapenem-resistant Acinetobacter baumannii
in intensive care unit patients: Risk factors for acquisition, infection and their consequences. J Hosp Infect 2007;65:204-11.
Bernards AT, Harinck HI, Dijkshoorn L, van der Reijden TJ, van den Broek PJ. Persistent Acinetobacter baumannii
? Look inside your medical equipment. Infect Control Hosp Epidemiol 2004;25:1002-4.
Tiwari V, Kapil A, Moganty RR. Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii
isolated from India. Microb Pathog 2012;53:81-6.
Tiwari V, Vashistt J, Kapil A, Moganty RR. Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii
with a reference strain. PLoS One 2012;7:e39451.
Alsultan AA, Hamouda A, Evans BA, Amyes SG. Acinetobacter baumannii
: Emergence of four strains with novel bla (OXA-51-like) genes in patients with diabetes mellitus. J Chemother 2009;21:290-5.
Dettori M, Piana A, Deriu MG, Lo Curto P, Cossu A, Musumeci R, et al.
Outbreak of multidrug-resistant Acinetobacter baumannii
in an intensive care unit. New Microbiol 2014;37:185-91.
Nageeb W, Kamel M, Zakaria S, Metwally L. Phenotypic characterization of Acinetobacter baumannii
isolates from intensive care units at a tertiary-care hospital in Egypt. East Mediterr Health J 2014;20:203-11.
Kempf M, Rolain JM. Emergence of resistance to carbapenems in Acinetobacter baumannii
in Europe: Clinical impact and therapeutic options. Int J Antimicrob Agents 2012;39:105-14.
Evans BA, Hamouda A, Abbasi SA, Khan FA, Amyes SG. High prevalence of unrelated multidrug-resistant Acinetobacter baumannii
isolates in Pakistani military hospitals. Int J Antimicrob Agents 2011;37:580-1.
Dent LL, Marshall DR, Pratap S, Hulette RB. Multidrug resistant Acinetobacter baumannii
: A descriptive study in a city hospital. BMC Infect Dis 2010;10:196.
Chang Y, Luan G, Xu Y, Wang Y, Shen M, Zhang C, et al.
Characterization of carbapenem-resistant Acinetobacter baumannii
isolates in a Chinese teaching hospital. Front Microbiol 2015;6:910.
Merino M, Poza M, Roca I, Barba MJ, Sousa MD, Vila J. et al
. Nosocomial outbreak of a multi-resistant Acinetobacter baumannii
expressing OXA-23 carbapenemase in Spain. Microb Drug Resist 2014;20:259-63.
Zowawi HM, Sartor AL, Sidjabat HE, Balkhy HH, Walsh TR, Al Johani SM, et al.
Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii
isolates in the Gulf Cooperation Council States: Dominance of OXA-23-type producers. J Clin Microbiol 2015;53:896-903.
El-Shazly S, Dashti A, Vali L, Bolaris M, Ibrahim AS. Molecular epidemiology and characterization of multiple drug-resistant (MDR) clinical isolates of Acinetobacter baumannii.
Int J Infect Dis 2015;41:42-9.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]