Detection of Carbapenem Resistant Acinetobacter: From Clinical Samples Naheed Afshan1*, Huda Fatima1 1Department of Microbiology, Jinnah University for Women, Karachi. ABSTRACT Acinetobacter has appeared from an organism of uncertain pathogenecity towards an infectious agent. Among nonfermenting bacterium A. baumannii is the second-most-commonly-isolated organisms in human. The fast intensify of their resistance to antibiotics, especially global emergence and extend of Acinetobacter strains resistant to carbapenem more restricted the therapeutic alternatives. In this study we evaluate the % of resistivity of Acinetobacter against carbapenem antibiotics at Jinnah University for Women, Karachi. Total 439 isolates of Acinetobacter were collected from different clinical samples of hospitalized patients from January to December 2013, identified by standard microbiological methods. Antibiograms were done on Mueller-Hinton agar plates with disk diffusion method (Kirby Bauer method). Disc tested: Meropenem (10 µg/disk). The results were interpreted according to the Guidelines of the Clinical and Laboratory Standards Institute. (CLSI). Among 439 samples, 300 (68.3%) samples were resistant to Meropenem and the remaining that is 139 (31.7%) showed sensitivity to the drugs. In underdeveloped countries including Pakistan the contentment of multidrug resistance and their dissemination in Acinetobacter species is not a simple task. While multiple drug resistance is increasing in this pathogen, and carbapenem conflict is quickly spreading which may become a major threat in future. So in Pakistan needs detail and organized data about carbapenem resistant Acinetobacter in order to understand the existence of Acinetobacter in our community and to manage almost certainly outbreaks because we have less information according to resistance trends of Acenetobacter. Keywords: Antibiogram, Carbapenem, Resistant, Outbreaks. INTRODUCTION Species of genus Acinetobacter are gram-negative belongs to the class Gammaproteobacteria. Species are non-motile, coccobacili in shape appear in pairs, oxidase-negative (Peleg et al., 2008). Acinetobacter consist of 27 authentically named and 11 unknown (genomic) species. Species names, including: A. baumannii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, and A. lwoffii (Manchanda et al., 2010). Widely dispersed in environment they can be alive on dry and moist surfaces, as well as in hospital surroundings (Maragakis and Perl, 2008). Patients in the intensive care unit (ICU), including burn patients, trauma patients, and patients requiring mechanical ventilation are mainly affected by Acinetobacter baumannii (Bassetti et al., 2008). Also some strains have been found from foodstuffs, water, and associate with low occurrence of allergies. Several species of Acinetobacter can cause serious infections in immunocompromised patients (Peleg et al., 2008). They also show comparatively wide scale of antibiotic resistance (Towner, 2009). Infections together with skin and wound added complication as well as bacteremia, and meningitis (Choi et al., 2008). The bacteria have the ability to colonize in medical utensils e.g. catheters (Doughari*Corresponding author: naheedafshan7@hotmail.com 09 Vol 5 (1), January 2014; 09-13 et al., 2011). Plasmid-borne markers which made the Acinetobacter resistant to various antibiotics are capable of transferring to new pathogenic bacteria through horizontal gene transfer (Juni, 1978). ‘MDR Acinetobacter spp.’ defined as the isolate resistant to at least three classes of antimicrobial agents — all penicillins and cephalosporin (including inhibitor combinations), fluroquinolones, and amino glycosides. ‘XDR Acinetobacter spp.’ that is resistant to the three classes of antimicrobials described above (MDR) and shall also be resistant to carbapenems (Kurcik-Trajkovska, 2009). The clinical isolates of Acinetobacter spp. were generally susceptible to gentamicin, minocycline, nalidixic acid, ampicillin, or carbenicillin, singly or in a combination therapy, throughout the early 1970s (Bergogne-Bérézin and Towner, 1996). But, since 1975, growing resistance started to appeare in almost all groups of drugs including the first and second generation cephalosporins. Primarily, they retained at least partial susceptibility against the third and fourth generation cephalosporins, fluoroquinolones, semi synthetic aminoglycosides, and carbapenems, with nearly 100% isolates holding susceptibility to imipenem (Vila et al., 2007). However, during late 1980s and 1990s, worldwide emergence and spread of Acinetobacter strains resistant to imipenem further limited the therapeutic alternatives. By the late 1990s, the only useful agents that fight many severe infections caused by Acinetobacter sp. were carbapenems (Cunha, 2013). Moreover, the therapeutic options are decreasing due to the emergence of carbapenem resistance in the strains of A. baumannii (SPL, 2011). Various mechanisms have been found to be accountable for the resistance to carbapenems in A. baumannii (CUH, 2013). The resistivity mechanismsusually fall into 3 categories: 1. antimicrobial-inactivating enzymes, 2. reduced access to bacterial targets, or 3. mutations that change targets or cellular functions (Camp and Tatum, 2010). Treatment is difficult for healthcare-acquired infection caused by A. baumannii resistant to imipenem (Smith et al., 2007). Carbapenems still represent the treatment of choice. A. baumannii is competent to grow at different temperature and pH because it does not have fastidious growth requirements. The versatile organism exploits a variety of both carbon and energy sources. These qualities explain the ability of Acinetobacter species to persist in either moist or dry conditions in the hospital environment, thereby contributing to transmission. This hardi- ness, combined with its intrinsic resistance to many antimicrobial agents, contributes to spread in the hospital setting. MATERIALS AND METHODS Setting: Department of Microbiology, Jinnah University for Women Karachi. Duration of study: From January 2013to December 2013 Sampling technique: Non-duplicate consecutive sampling. Inclusion Criteria: All Acinetobacter colonies isolated from different clinical samples of patients. Exclusion Criteria: Sample showing no growth or growth of gram positive bacteria, growth of gram negative bacteria other than Acinetobacter and yeast. Repeat and duplicate samples from the same patient were also being excluded. Study design: Descriptive study. 10 Vol 5 (1), January 2014; 09-13 Figure 1: Outbreaks of Acinetobacter in United States between 2002 and 2007 (Chuang et al., 2011). Data collection: All clinical samples were collected in sterilized container according to samples from patients of different hospitals and platted right after the collection. Identification will be taken by standard microbiological methods. Inoculation of clinical samples was done on standard media such as sheep blood agar (SBA) Mackonkey agar and Chocolate Agar. Antibiograms were done on Mueller-Hinton agar plates with disk diffusion method according to Kirby Bauer method. Disc tested: Meropenem (10 µg/disk) (Oxoid Ltd., England) The results was interpreted according to the Guidelines of the Clinical and Laboratory Standards Institute (CLSI). RESULTS DISCUSSION We have performed our studies on 439 isolates. We hereby observed the sensitivity and resistivity pattern of Meropenem on different samples including pus, urine, tracheal aspirates, sputum, blood and body fluids which were loaded with Acinetobacter. We observed that among 439 samples, 295(67%) samples were resistant to Meropenem the remaining i.e. 144(33%) showed sensitivity to the drug. Out of 439 samples 195 were tracheal aspirates, 34 were blood samples, 30 were urine, 101 were pus and swabs, 41 were sputum samples and 38 were fluid samples. This increased resistivity of Acinetobacter is considered as an important health problem due to considerable clinical impact of this resistivity on the management of health care associated infections. In intensive care unit, the serious concern of high morbidity and high mortality rates of A.baumanii is their nosocomial outbreaks. Clinical threats scored by acinetobacter are its adaptation to the environment, versatile metabolism and its ability to develop resistance against antibiotics that are used in clinical settings. This danger has caused an noticeable and alarming decline in the available chemotherapeutic resources, that includes drugs like, carbapenem antibiotics that inhibits peptidoglycan biosynthesis that was considered the first-rate standard for Acinetobacter treatment until recently (Guerrero et al., 2010). Infections due to Acinetobacter frequently involve organ systems that have a high fluid content (eg, respiratory tract, CSF, peritoneal fluid, urinary tract), manifesting as nosocomial pneumonia, associated with continuous ambulatory peritoneal dialysis (CAPD), or catheter-associated bacteruria. The presence of Acinetobacter isolates in respiratory secretions in incubated patients nearly always represents colonization. Acinetobacter pneumonia occur in outbreaks and are usually associated with colonized respiratory-support equipment or fluids. Nosocomial meningitis may occur in colonized neurosurgical patients with external ventricular Figure 3: Sensitivity & resistivity % of meropenem among different clinical samples. 11 Vol 5 (1), January 2014; 09-13 Figure 2: Sensitivity & resistivity % of meropenem of total samples. Table I: Susceptibity pattern of Acinetobacter sp. from different samples. drainage tubes. A. baumannii is a multiresistant aerobic gram- negative bacillus sensitive to relatively few antibiotics. Multidrug-resistant Acinetobacter is not a new or emerging phenomenon, but A. baumannii has always been an organism inherently resistant to multiple antibiotics. Since the past decade, antimicrobial resistance among Acinetobacter sp. is a rising concern. The species are equipped with extensive antimicrobial resistance due to the presence of the porin channels, efflux mechanisms and the non static behaviour of the bacteria in hot and humid conditions (Davies and Rubin, 2007). CONCLUSION In underdeveloped countries including Pakistan , india and Bangladesh the contentment of multidrug resistance and their dissemination in Acinetobacter sp. is not a simple task. While multiple drug resistance is increasing in this pathogen, and carbapenem conflict is quickly spreading which become a major threats in future because patient-to-patient transmission in hospitals through contaminated hands of healthcare workers and fomites is the main factors w h i c h i n c r e a s e s t h e s p r e a d i n g o f M D R Acenetobacter and cure should be followed. The increased resistivity of the pattern of the organism has become a serious threat for health in Pakistan. REFERENCES Bassetti M, Righi E, et.al 2008. Drug Treatment for Multidrug-resistant Acinetobacter baumannii Infections. Future Microbiol.;3(6):649-660. Bergogne-Bérézin E and K J Towner.1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological f e a t u r e s . C l i n . M i c r o b i o l . R e v. , 9 ( 2 ) : 1 4 8 . Camp C, Tatum OL.2010. A Review of Acinetobacter baumannii as a Highly Successful Pathogen in Times of War Callie Camp. Lab Med.;41(11):649-657 Choi CH, Jun Sik Lee1, et.al . 2008. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiology, 8:216 doi:10.1186/1471-2180-8-216 Chuang YC et.al.2011. Influence of genospecies of Acinetobacter baumannii complex on clinical outcomes of patients with acinetobacter bacteremia. Clin Infect Dis, 52(3):352-360. CUH. 2013. Acinetobacter. Cambridge University Hospitals. Infectious Control. http://www.cuh.org. uk/cms/addenbrookes-hospital/infection-control/well- known-infections-faqs/acinetobacter C u n h a B A . 2 0 1 3 . A c i n e t o b a c t e r. http:// e m e d i c i n e . m e d s c a p e . c o m / a r t i c l e / 2 3 6 8 9 1 - overview#a0199 Davies JC, Rubin BK.2007. Emerging and Unusual Gram-Negative Infections in Cystic Fibrosis. Semin. R e s p i r. C r i t . C a r e M e d . , 2 8 ( 3 ) : 3 1 2 - 3 2 1 . Doughari HJ, Lois PA et.al. 2011. The Ecology, Biology and Pathogenesis of Acinetobacter spp. An Overview. Microbes Environ. 26(2). doi:10.1264/ jsme2.ME10179 Guerrero DM et.al.2010. Acinetobacter baumannii- Associated Skin and Soft Tissue Infections: Recognizing a Broadening Spectrum of Disease. Surg. Infect. (Larchmt), 11(1): 49–57. Juni, E. 1978. Genetics and Physiology of Acinetobacter. Ann. Rev. Microbiol. 32:349-371. Kurcik-Trajkovska B. 2009. Acinetobacter spp. – A Serious Enemy Threatening Hospitals. Macedonian J o u r n a l o f M e d i c a l S c i e n c e s , 2 ( 2 ) : X X - XX.doi:10.3889/MJMS.1857-5773.2009.0043 12 Vol 5 (1), January 2014; 09-13 13 Vol 5 (1), January 2014; 09-13 Manchanda V, et.al. 2010. Multidrug Resistant Acinetobacter. J Glob Infect Dis., 2(3): 291–304. Maragakis LL, Perl TM. 2008. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis., 46(8):1254- 63. doi: 10.1086/529198. Peleg AY, Harald Seifert, et al .2008. Acinetobacter baumannii: Emergence of a Successful Pathogen. Clin Microbiol Rev. July; 21(3): 538–582 Smith, MG et.al. 2007. New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. doi:10.1101/gad.1510307 Genes & Dev.. 21: 601-614 SPL. 2011. Acinetobacter Fact Sheet. http://www. specialpathogenslab.com/SPL-Advantage/FactSheet - Acinetobacter. pdf Towner KJ. 2009 ‘’ Acinetobacter: An old friend, but a new enemy’’. J Hosp Infect.; 73: 355-363. Vila, J.; Marti, S.; Sanchez-Cespedes, J. 2007. Porins, efflux pumps and multidrug resistance in Acinetobacter baumannii. 59(6):1210-1215. Xiao YH et.al.2011. Epidemiology and characteristics of antimicrobial resistance in China. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy, 14(4–5):236-250. Yang YS et.al.2013. Comparison between bacteremia caused by carbapenem resistant Acinetobacter baumannii and Acinetobacter nosocomialis. BMC Infectious Diseases, 13:311 doi:10.1186/1471-2334- 13-311