2612 Page. *Yusuf, I 1., Rabiu A.T 1., Haruna, M 2. and Abdullahi, S. A 3. Phone:

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1 Carbapenem-Resistant Enterobacteriaceae (CRE) in Intensive Care Units and Surgical Wards of hospitals with no history of carbapenem usage in Kano, North West Nigeria *Yusuf, I 1., Rabiu A.T 1., Haruna, M 2. and Abdullahi, S. A 3 1Department of Microbiology, Faculty of Science, Bayero University, P.M.B 3011, Kano-Nigeria 2Department of Biology, Kano University of Science and Technology, Wudil, Kano, Nigeria 3Pathology Department, Nuhu Bamalli Maternity Hospital, Kano -Nigeria Phone: Abstract: Study was conducted to determine carbapenem resistant Enterobacteriaceae (CRE) in intensive care units (ICUs) and surgical wards (SW) of tertiary health care centers in Kano, North West Nigeria. There was no history of carbapenem usage in the hospitals studied but routine antibiotic susceptibility tests in the hospitals have showed increased resistance of Escherichia coli and Klebsiella pneumoniae to the antibiotics. In addition, the pathogens were screened for susceptibility to colistin and tigecycline. Bacteria isolated from urine, cathertips, blood stream and cerebrospinal fluids of patients admitted into ICUs or SW for at least 7 days were tested for susceptibility to imipenem (IMP) 30µg and meropenem (MEM) 10µg using disc diffusion method according to Clinical Laboratory Standards Institute (CLSI) 2012 break points. Carbapenemase and Metallo-β-lactamase (MBL) production were detected phenotypically by Modified Hodge Test (MHT) and Combine Disc Test (CDT) respectively. Out of the 200 bacterial isolates recovered from the study, E. coli was 42.0%, K. pneumoniae 30.5%, Proteus sp 16.0% and Ps. aeruginosa (11%). Resistance to IMP and MEM (zone diameter 19mm) was 10.5% (21/200) and 12.5% (25/200) respectively. Carbapenemase production among the pathogens was 34.5% (highest of its kind in Kano). Some of the pathogens with intermediate sensitivity to either IMP or MEM also produce carbapenemase. About 14.4% of the carbapenemase producers produce MBL. All the isolates were susceptible to colistin and tigecycline, with exception of 3 isolates (2 E. coli and 1 K. pneumoniae) that are totally resistant to colistin and tigecycline. Interestingly, none of the 4 isolates produced carbapenemase. This showed that carbapenem resistance was in increase in hospitals where carbapenems were not in use and the resistances were mediated by carbapenemase especially by MBL production and can be treated with colistin and tigecycline. Keyword: carbapenem, colistin, tigecycline, carbapenem-resistant-enterobacteriaceae, Nigeria INTRODUCTION Carbapenems are one of the antibiotics of last resort for many bacterial infections such as E. coli and K. pneumoniae producing AmpC and extended spectrum ß-lactamase (ESBL) but carbapenemase enzyme which have versatile hydrolytic capacities have the ability to hydrolyze penicillins, cephalosporins, monobactams and carbapenems (Fernando et al., 2009). *Corresponding author: iyusuf.bio@buk.edu.ng; *Yusuf, I 1. Copyright 2015 Nigerian Society for Microbiology Nigerian Journal of Microbiology 2015, 27(1): Published online at The broad-spectrum β-lactam antibiotics, carbapenems, were introduced in 1985 and have been for years the most important agents for the treatment of infections caused by multidrug-resistant bacteria. Carbapenem resistance is now observed increasingly worldwide, and constitutes a sentinel event for emerging antimicrobial resistance (Poirel and Nordman, 2006; Peleg et al., 2008). Multidrug-resistant isolates have been reported increasingly during the last decade in many parts of Nigeria (Aibinu et al., 2007; Nkang et al., 2009; Ewurum et al., 2011; 2612 Page

2 Yusuf et al., 2012), other African countries (Andrew, 2009) probably as a consequence of extensive misuse of antimicrobial agents in the region and in western countries (Raffaele et al., 2009) due to extensive use of antibiotics. Infections caused by organisms producing such enzymes have resulted in poor outcomes, reduced rate of clinical and microbiological responses, longer hospital stays and greater hospital expenses (Pertason et al., 2004). Physical contact is the most likely mode of transmission and the gastrointestinal tract of colonized or infected patients is the most frequent reservoir while transient carriage of bacteria on the hands of healthcare workers may lead to transmission to patients (Pertason et al., 2004). The pathogens have been reported to resist treatment with first and second line antibiotics but are susceptible only to colistin and, less consistently, tigecycline (Kummarasamy et al., 2010). Acquired carbapenemases are a structurally diverse group of beta lactamase that, in most cases, hydrolyse not only carbapenems, but also oxyiminocephalosporins and cephamycins (Struelens et al., 2010). For years, clinical isolates of Enterobacteriacea and other community Gram negative bacteria have begun to acquire different types of carbapenemases. In Europe, VIM-type MBLs and the socalled K. pneumoniae carbapenemase (KPC) are the most frequently isolated carbapenemases, although K. pneumoniae producing the class D OXA-48 carbapenemase is prevalent in Turkey (Nordman et al., 2009). Reports of such serotypes were scanty in Nigeria, but phenotypic detection of carbapenem resistance among clinical isolates in some hospitals in Kano, North west Nigeria were reported by Yusuf et al. in 2011 with low prevalence. Nevertheless carbapenemaseproducing strains of K. pneumoniae harbouring either blavim or blakpc have been the cause of country-wide epidemics of healthcare associated infections in Greece, Israel, the United States (US), several Latin American countries and China, and of local outbreaks in Poland and Italy (Nordmann et al., 2009). The spread of these resistant bacteria in hospitals all over the world, conferring multiple antibiotic resistances in the treatment and management of life threatening infections, coupled with the fact that risen incidences of multiple antibiotic resistances were observed in many hospitals in the region, make the study necessary. The routine susceptibility tests performed by clinical laboratories in Nigeria, fail to detect bacteria harboring these enzymes, which have led to inappropriate and unsuccessful therapy of patient and unnecessary use of drugs. This study was therefore aimed at determining the prevalence of CRE in ICUs and SW of hospitals with no history of carbapenem usage and to test the susceptibility of the CRE to colistin and tigecycline. MATERIALS AND METHODS Sample Collection The isolates from patients admitted into the ICUs and SW of two specialist hospitals in Kano, North West Nigeria, were used for the study after obtaining an ethical clearance from the respective Hospitals ethical committees. Samples of such patients were carefully monitored from collection, transportation to laboratory and isolation of pathogens with the help of nurses in the units and clinical laboratory scientists in the microbiology laboratory. Specimens of blood, urine, cather-tips, wound swabs and few stool were used and isolates of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, P. vulgaris and Pseudomonas aeruginosa were isolated over a period of four months. Screening and confirmatory test for carbapenemase All the isolates were phenotypically screened for carbapenemase production according to Clinical and Laboratory Standard Institute (CLSI, 2012) directives. We used two standard carbapenem discs i.e Page

3 imipenem (30 µg) and meropenem (10 µg), (Oxoid UK) using disc diffusion test. Any isolate that is either totally resistant or showed reduced susceptibility with zones of inhibition of less than 23 mm and 21 mm for MEM and IMP respectively were assumed to be potential carbapenemase producers and were subjected to further tests for confirmation. Confirmatory Tests Modified Hodge Test for Carbapenemase The modified Hodge method described by the CLSI, 2012 was employed where a 1/10 dilution of an inoculum of a control strain of E. coli ATCC 25922, adjusted to a 0.5 Mc Farland turbidity standards was used to inoculated the surface of a plate of Mueller Hinton Agar (MHA) (Oxoid, UK). The plates were then allowed to stand for 10 minutes at room temperature. Two discs one with IPM (30µg), and other with MEM (10µg), were placed onto the agar plate. Subsequently a 10 µl pipette was used to pick three to five colonies of each test organism, grown overnight on an agar plate and inoculated onto the plate in a straight line from the edge of one disk to another. The plates were then incubated at 37 C for 18 h. Screening of carbapenemase producers for metallo ß-lactamases (MBLs) EDTA-disc synergy test was used as described by Walsh et al.2005 and CLSI All the ICUs obtained isolates were streaked on the surface of the MHA using a sterile wire loop. Two MEM discs, one containing EDTA and the other without EDTA were placed on the surface of each of the MHA using a sterile forceps at a distance of about 30 mm apart. The EDTA disc was prepared by adding 250 µl of neutral EDTA to MEM. The plates were incubated for 24 h at 37 C. Zones of inhibition were recorded after 24 h of incubation. Control disc containing EDTA alone was used to determine the activity of the EDTA to ensure that it does not cause false-positive results by inhibiting the test isolates. Organisms that showed differences in diameter of zone of inhibition by 4 mm on the MEM containing EDTA and MEM alone are confirmed for the production of MBLs. Antimicrobial Susceptibility Test An antibiotic susceptibility test was performed on both carbapenemase and MBL producers to colistin and tigecycline by disc diffusion method. The zone diameters of each of the antibiotics were interpreted as per CLSI 2012 recommendations. Briefly, a 0.5 McFarland suspension of the test organisms were inoculated on the surface of MHA plates. The plates were allowed to stand for 10 minutes at room temperature after which two discs, one of colistin (10 µg), and other tigecycline (15 µg)(oxoid UK), were placed onto the agar plate. Isolates producing a diameter of >14 mm and >21 mm for colistin and tigecycline respectively were considered susceptible. E. coli ATCC was used as the control strain for the antimicrobial susceptibility testing. RESULTS Out of the total 319 clinical bacterial isolates obtained from the ICUs and SW in the hospitals were carbapenems were not in use, carbapenem resistance due to carbapenemase and MBL was 28.2 and 8.9% respectively. The carbapenemase production cut across all the isolates with less prevalence in P. mirabilis (15.7%). Interestingly, all the MBL producers are obtained from carbapenemase producers with the exception of P. vulgaris which did not produce MBLs (Table 1). Carbapenem resistant isolates were more prevalent in blood, catheter tips and urine but surprisingly, no carbapenemase and MBLs producers in the few isolates from stool sample. Carbapenemase production in wound swab isolates are serine based with very low MBLs (1.1%) (Table 2). Susceptibility of both carbapenemase and MBLs producers to colistin and tigecycline is high in all the 2614 Page

4 isolates except Ps. aeruginosa with moderate susceptibility. Table 1 Incidence of carbapenemase and MBL producers among ICUs and SW isolates Bacterial Species No. of isolates No. of carbapenemase No. of MBL Producers screened producers E. coli (30.8) 09 (8.4) K. pneumoniae (29.9) 10 (10.3) P. mirabilis (23.4) 05 (10.6) P. vulgaris (15.7) 00 (0.0) Ps. aeruginosa (28.3) 04 (8.7) Total (28.2) 28 (8.9) NB: Values in parenthesis are percentages Table 2: Distribution of carbapenemase and MBL producing ICUs and SW isolates among clinical samples Clinical samples No. of isolates screened No. of carbapenemase producers No. of MBL producers Blood 36 11(30.6) 06 (16.6) Catheter tip (37.5) 04 (25.0) Stool (0.0) 00 (0.0) Urine (34.5) 17 (10.6) Wound swab (17.2) 01 (1.1) Total (28.1) 28 (8.9) NB: Values in parenthesis are percentages Table 3: Susceptibility of carbapenemase producing clinical bacterial isolates to colistin and tigecycline. Bacterial Species Antimicrobial Susceptibility Colistin Tigecycline No. susceptible (%) No. susceptible (%) E. coli (33) 31 (93.9) 28 (84.8) K. pneumoniae (29) 27 (93.1) 23 (85.1) P. mirabilis (11) 11 (100) 11 (100) P. vulgaris (3) 03 (100) 01(33.3) Ps. aeruginosa (13) 07 (53.8) 10 (76.9) 2615 P age

5 Table 4 Susceptibility of MBL producing clinical bacterial isolates to colistin and tigecycline. Bacterial Species Antimicrobial Susceptibility Colistin Tigecycline No. susceptible (%) No. susceptible (%) E. coli (9) 09 (100.0) 09 (100.0) K. pneumoniae (10) 05 (50.0) 07 (70.0) P. mirabilis (5) 04 (80.0) 05 (100) Ps. aeruginosa (4) 02 (50.0) 02 (50.0) DISCUSSION Antibiotic resistance among clinical bacterial isolates in Nigerian hospitals is increasing yearly. Only few hospitals in Nigeria have sustainable antibiotic resistance and infection control units and only few data are available in accessible forms regarding antibiotic resistance in Nigerian hospitals. Some hospitals have reported various prevalence of ESBL, AmpC and MBLs and carbapenemase production in many states of Nigeria. For instance Aibinu et al., 2007 and Enwuru et al., 2011 have reported the incidence of ESBL and MBLs among some isolates in Lagos Nigeria south West Nigeria. Similarly reports of ESBLs AmpC, carbapenemase and MBLs have been reported by Yusha u et al., 2007, Yusuf et al., 2011, Yusuf et al., 2012 in Kano North West geographical zone of Nigeria. The menace of antibiotic resistance is a global problem but the spectrum and extent of the problem may vary from country to country or region to region. Data have shown some countries are taking suitable efforts to confront the issue but in Nigeria, attentions are not paid to these problems by many hospitals because the awareness among the medical practitioners is low. As a result of this negligence, carbapenems antibiotics which are one of the antibiotics of last resort for many bacterial infections caused by E. coli, Ps. aeruginosa and K. pneumoniae producing AmpC and ESBL have developed resistance to carbapenems despite their unavailability in many hospitals we studied. This is amazing, but reports by Andrew, (2012) have shown that about 90.1% individuals in Africa seek care outside the home, and of these, 94.7% take medicines and 36.2% receive antibiotics. Of all those who receive antibiotics, 31.7% do not receive prescription from doctor and about 26.4% obtain their antibiotic from illegal dispensers. In this study, carbapenemase production is evident among E. coli, K. pneumoniae, P. mirabilis and Ps. aeruginosa isolated from ICUs and SW of the hospitals. The source of these CRE is not known because data regarding previous treatments and hospitals visited in the past is not available. Although previous studies have shown occurrence of carbapenemase in non ICUs isolates in a tertiary hospital were carbapenems are used in some cases (Yusuf et al., 2012). Antibiotic resistance has no boarder to cross especially in settings where proper infection control is not in practice. Resistance to carbapenems in Nigeria is gradually increasing. In 2007, Aibinu et al. reported 95.9% susceptibility of Ps. aeruginosa to both IMP and MEM, while in 2008, E. coli and K. pneumoniae were 100% susceptible to IMP in the same state (Enwuru et al., 2011). In 2011, resistance of E. coli, K. pneumoniae and Ps. aeruginosa to IMP ranged from 6-9% in Kano North west Nigeria (Yusuf et al., 2012). Multiple factors have contributed to increasing resistance to antibiotics especially in developing countries such as Nigeria. Common ones have been attributed to uncontrolled sales 2616 Page

6 of antibiotics in markets, streets, buses and availability of counterfeit drugs in circulation and free access to antibiotics. These have resulted in emergence of different multi drug resistant pathogens and ease its smooth transmission from hospitals to community. However, recently, government in the affected studying state in its effort to reduce misuse of antibiotics have taken a step to minimize easy access to antibiotics and to make sure antibiotics are handled by professionals by banning the sales of drugs in an open market in the state, the decision that face total rejection by the marketers. The occurrence of both MBL and serine based carbapenemase in pathogens from these hospitalized patients will ultimately lead to treatment failure especially if penicillins, 3rd and 4th generation cephalosporins are used. In addition, it will reduce treatment options for the clinicians as well as higher treatment cost resulting from longer hospital stay and need for more costly and potential toxic medications such as colistin and tigecycline. What will be the next option for treating CRE in these hospitals if carbapenems which are not in use in the hospitals are gradually becoming useless? Some researchers have reported the susceptibility of MBLs such as New Delhi Metallo beta lactamase (NDM-1) to colistin and less consistently tigecycline (Young et al., 2009 and Kumarasamy et al., 2010). This study agreed with such reports in which majority of carbapenemase and MBL producing pathogens are susceptible to colistin and tigecycline. The few exceptions are Ps. aeruginosa which showed moderate susceptibility to both and some strains of serine based carbapenemase producing P. vulgaris which are resistant to tigecycline. There are 3 strains of CRE (2 E. coli and 1 K. pneumoniae) that are totally resistant to colistin and tigecycline. Interestingly, none of the 3 isolates produces carbapenemase. This showed that these antibiotics are promising in the treatment of life threatening infections caused by CRE in ICUs and SW. Both carbapenemase and MBLs producers were obtained more from blood, urine and cather tips of the patients, showing the great chance of transmission attributed to factors like improper use of syringes or needles, inadequate disinfection of skin of prolong hospital stayed patients during phlebotomy or transfusion and poor hand washing technique among health practitioners. However, a lower incidence was recorded for surgical wound pathogens i.e. 17.1% and 1.1% for carbapenemase and MBLs respectively. CONCLUSION The study has shown increasing resistance of pathogens obtained from patients admitted into the ICUs and SW of many hospitals that are not using carbapenems. The majority of carbapenems resistances are mediated by both serine and metallo base carbapenemase. Activities of health care workers in catheterization and phlebotomy are major factors in their acquisition and transmission. Susceptibility of the pathogens to colistin and tigecycline was promising and could be drug of choice in life threatening cases in view of their toxicity. REFERENCES Aibinu, I., Tochukwu, N. and Tolu, O. (2007). Occurrence of ESBL and MBL in clinical isolates of Pseudomonas aeruginosa from Lagos, Nigeria. Journal of American Science, 3(4): Andrew, N.K. (2012). A situational analysis of antimicrobial drug resistance in Africa: are we losing the battle? Ethiopian Journal of Health Science, 22(2): Clinical and Laboratory Standards Institute. (2012). Performance standards for antimicrobial susceptibility testing: 22 nd Informational supplement. CLSI document M100-S22. Clinical and 2617 Page

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