July 2008.indd Extended-spectrum β-lactamase (ESBL) in Omani Children Study of prevalence, risk factors and clinical outcomes at Sultan Qaboos University Hospital, Sultanate of Oman Zakariya Al Muharrmi,1 *Akbar M Rafay,1 Abdullah Balkhair,2 Salem Al-Tamemi,3 Ali Al Mawali,4 Hilal Al Sadiri4 في (ESBL) املديد الطيف ذي البيتاالكتاميز التي تفرز البكتيريا األطفال العمانيني جامعة مستشفى في السريرية والنتائج تِطار لُ االِخْ وامِ وعَ االنتشار دراسة عمان سلطنة قابوس، السلطان السديري هالل ، علي املعولي ، التميمي سالم بلخير ، عبداهللا ، رافع أكبر احملرّمي، زكريا الى تهدف ــة الدراس هذه . ــب املناس اختيار املضاد احليوي في صعوبات ، وهذا يضع العالم في ــكلة متنامية مش البكتريا مقاومة امللخص: الهدف: شهر إلى شهر يناير من قابوس السلطان جامعة مبستشفى في قسم األطفال (ESBL) املديد الطيف البيتاالكتاميز ذي تفرز التي تقييم البكتيريا عند االختطار عوامل مت حتليل . قسم األطفال في الراقدين األطفال البكتيريا عند تلك ــة ودراس مت حتديد ــهرا 12 ش الطريقة: خالل .2005 ــمبر ديس الطيف ذي البيتاالكتاميز تفرز الرئوية الكليبسيال 16.6 %) من و( املعوية االشريشيا من (13.3% ): النتائج . البكتريا بتلك أصيبوا الذين املرضى استخدام هو البكتيريا بهذه اإلصابة إلى أدت التي االختطار عوامل وأهم .(42.6% ) الدم ومن ، البول من (46.2 %) جاءت هذه البكتريا غالبية . املديد ــية حساس كانت .(84.6% ) أنثى املريض وكون ،(92.3% ) ــديد واملرض الش ــفى املستش في الطويل من الترقيد %100)، وكال ) ــة احليوي ــادات املض لم ــبورين. سيفالوس أماينو- ــي- األوكس ضد (100 %) مقاومة ــا أبدى جميعها (92 %) ، بينم ــني لألميكاس و ، (100% ) ــم للكاربابين ــا البكتري النايتروفرونتوين. اجلنتاميسني مع النايتروفرونتوين، مع أميكاسني تازوباكتم، البيبراسيلني- مع التالية: األميكاسني ضد التوليفات مناعة أية تسجل للمضادات ــبق املس وأن التعرض ، املرضى العمانيني األطفال ــكلة كبيرة عند مش املديد الطيف ذي البيتاالكتاميز تفرز البكتيريا التي متثل اخلالصة: االستعاضة وميكن ، الكاربابينم هو البكتريا لهذه األمثل العالج البكتيريا. هذه انتشار لتقليل بها التحكم ميكن هي عوامل الطويل والترقيد احليوية النايتروفرونتوين. اجلنتاميسني مع النايتروفرونتوين، مع أميكاسني تازوباكتم، البيبراسيلني- مع التالية: األميكاسني بالتوليفات عنه عمان. احليوية، املضادات االختطار، عوامل الرئوية، الكليبسيال ، املعوية االشريشيا املديد، الطيف ذي الكلمات: البيتاالكتاميز مفتاح Departments of 1Microbiology & Immunology, 2Medicine, 3Child Health, Sultan Qaboos University Hospital, Muscat, Oman; 4Medical Student, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, Oman *To whom correspondence should be addressed. Email: akbarrafay@hotmail.com SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL JULY 2008, VOLUME 8, ISSUE 2, P. 171-177 SULTAN QABOOS UNIVERSITY© SUBMITTED - 3RD OCTOBER 2007 ACCEPTED - 31ST MARCH 2008 ABSTRACT Objectives: Antimicrobial resistance is a growing problem worldwide, which imposes difficulties in the selection of appropriate empirical antimicrobial therapy. This study evaluated extended-spectrum β-lactamase (ESBL) isolates in 2005 in The Department of Child Health at Sultan Qaboos University Hospital (SQUH), Oman. Methods: During the 2 month period from January 2005 to December 2005, ESBL isolates from paediatrics inpatients were identified and analysed. Risk factors for the patients who grew ESBLs were analysed. Results: 3.3% of E. coli and 6.6% of Klebsiella pneumoniae isolated were ESBL producers. Most of the ESBLs were from urine (46.2%) and blood (42.6%). The main risk factors for ESBL in these children were previous exposure to antimicrobials (00%), prolonged hospital stay, severe illness (92.3%) and female gender (84.6%). Sensitivity of 00% was observed to carbapenems whereas 92% of the isolates were susceptible to amikacin. The oximino-cephalosporins were 00% resistant. Klebsiella pneumoniae were 00% resistant to piperacillin-tazobactam and nitrofurantoin. E. coli was 00% resistant to trimethoprim-sulfam- ethoxazole and ciprofloxacin. No resistance was recorded for the following combinations: amikacin plus piperacillin-tazobactam, amikacin plus nitrofurantoin and gentamicin plus nitrofurantoin. Conclusion: ESBL-producing organisms are becoming a major problem in Omani children. Exposure to antimicrobials and long admissions are modifiable risk factors that should be targeted for better control. Carbapenems are the most sensitive and reliable treatment options for infections caused by ESBLs. Amikacin plus piperacillin-tazobactam or nitrofurantoin are good alternatives. Keywords: Extended-spectrum β-lactamase; Escherichia coli; Klebsiella pneumonia; Anti-infective agents; Risk factors; Oman. C L I N I C A L A N D B A S I C R E S E A R C H Z A K A R I YA A L M U H A R R M I , A K B A R M R A FAY, A B D U L L A H B A L K H A I R , S A L E M A L -TA M E M I , A L I A L M AWA L I A N D H I L A L A L S A D I R I 172 ANTIMICROBIAL RESISTANCE IS A GROWING problem worldwide, which imposes difficul-ties in the selection of appropriate empirical antimicrobial therapy. Since the first extended-spec- trum β-lactamase (ESBL) producing Klebsiella pneu- moniae were discovered in Western Europe in the mid-1980s, the ESBL producing Enterobacteriacea became the focus of many scientific research stud- ies and investigations.1-5 ESBL are enzymes belong- ing to either class A or class D β-lactamases. Class A ESBLs belong to three types: SHV with more than 50 varieties currently recognized on the basis of unique combinations of aminoacid replacements; TEM with more than 130 TEM enzymes currently recognized; and CTX-M with more than 40 CTX-M enzymes cur- rently known.6 Other uncommon class A ESBLs are BES-1, GES-1, GES-2, IBC-1, IBC-2, PER-1, SFO-1, TLA-1, VEB-1 and VEB-2.6 There are also at least twelve ESBLs belonging to the OXA type (class D).6 ESBLs are plasmid-mediated, and their potential for transfer makes it increasingly difficult to control and treat these organisms effectively.7 As of 25 January 2005, there were 138 TEM- (TEM-1 to TEM-139) and 62 SHV-types (SHV-1 to SHV-63) of β-lactamases, mostly found in K. pneumoniae and E. coli strains.8 These mutant enzymes were termed ‘Extended-Spec- trum β-Lactamase’ by Philippon et al9 in 1989. ESBLs hydrolyse extended spectrum cephalosporins with an oxyimino side chain.10 These cephalosporins include cefotaxime, ceftriaxone and ceftazidime, as well as the oxyimino-monobactam aztreonam. In addition, ESBL- producing organisms are frequently resistant to many other classes of antibiotics, including fluoroquinolo- nes, the monobactam aztreonam, while resistance to trimethoprim–sulfamethoxazole and aminoglycosides is frequently co-transferred on the same plasmid.9, 11- 14 ESBLs are sensitive to cephamycins (cefoxitin, ce- fotetan) and carbapenems. ESBL-producing organ- isms are poorly responsive to treatment with wide spectrum cephalosporins such as ceftazidime and cefepime.7, 15 ESBL-producing organisms are difficult to differentiate from AmpC β-lactamase-producing Enterobacteriacea; however, most ESBL producers are generally susceptible to cephamycins (e.g. cefoxitin) in vitro. ESBLs are plasmid-mediated while AmpC β- lactamase enzymes are located on the chromosomes of Enterobacter sp, Citrobacter freundii, Morganella morganii, Serratia marcescens, and Pseudomonas aer- uginosa. The appearance of similar plasmid-mediated β-lactamases in K. pneumoniae and E. coli raises con- cerns over the spread of resistance, 7 which will further increase the difficulties of phenotypically identifying ß-lactamases.16-21 There are many precipitating factors for selection of ESBL producing organisms. These include the in- creasing use of oxyimino-β-lactams such as ceftazi- dime, cefotaxime and ceftriaxone. Other risk factors for the acquisition of ESBLs include presence of intra- vascular catheters; emergency intra-abdominal sur- gery; a gastrostomy or jejunostomy tube; gastrointes- tinal colonisation; length of hospital or intensive care unit stay; prior antibiotics (including third-generation cephalosporins); prior nursing home stay; severity of illness; presence of a urinary catheter and ventilator assistance.22 The problem of ESBL production is still rela- tively unappreciated by most clinicians.23, 24 This may be due in part to difficulty in laboratory identi- fication of ESBLs and misreporting them as sensitive organisms.15, 25-27 Many ESBL-producing isolates are not always phenotypically resistant to all oximino-ce- phalosporins; however, patients suffering from infec- tions caused by ESBL-producing organisms are at risk of treatment failure if treated with one of the oximino- cephalosporins.9, 28, 29 Therefore, it is imperative for the clinical microbiology laboratory to identify isolates that possess increased minimum inhibitory concentra- tions (MICs) (≥ 2 µg ⁄ mL) to oximino-cephalosporins, even though they may be equal to or below the suscep- tibility breakpoint (MIC ≤ 8 µg ⁄ mL).9 The rate of ESBL varies from country to country. The prevalence of ESBLs in the UK in 2002 was 7.4%.30 In Europe, the prevalence of ESBL producing E.coli is 10.8% while K. pneumoniae is 13.6%.31 In the USA, the prevalence of ESBL producing E.coli is 1.4% while K. pneumoniae is 4.4% 31. The prevalence of ESBL at Sul- tan Qaboos University Hospital, (SQUH), Oman is not yet known. We have analysed the sensitivity and dis- tribution of some ESBL isolates in SQUH previously32 without studying the prevalence rate of ESBL in SQUH as a whole, or in individual departments. In this arti- cle, we are reporting the prevalence of ESBL isolates in paediatric patients admitted to SQUH with an analy- sis of the risk factors and clinical outcomes of ESBLs infections. E X T E N D E D - S P E C T R U M β - L A C TA M A S E ( E S B L ) I N O M A N I C H I L D R E N 173 M E T H O D S SQUH is a 500-bed tertiary and teaching hospital cov- ering all major medical specialties. It is located on the campus of Sultan Qaboos University in Muscat, Oman. The Department of Child Health occupies three differ- ent wards. Each ward accommodates 24 beds of which 4 beds are for isolation. All specimens received from Department of Child Health from January-December 2005 were properly processed to identify ESBLs. Initially, the isolates were screened by a commercial system (Phoenix Identifica- tion and Susceptibility System from Becton Dickinson) for ESBL production. The positive results were further confirmed using the Clinical and Laboratory Stand- ards Institute (CLSI) approved double-disk diffusion method14, which is based on a synergistic increase of inhibition zone of ceftazidime and cefotaxime when they are combined with clavulanate. The test is consid- ered positive when the increase of the inhibition zone is (>/= 5 mm).9, 33, 34 Susceptibility results were recorded for the fol- lowing antimicrobials using the Phoenix Identifica- tion and Susceptibility System: gentamicin, amikacin, imipenem, meropenem, cefotaxime, ceftazidime, cefepime, ciprofloxacin, piperacillin-tazobactam, tri- methoprim/sulfamethoxazole and nitrofurantoin. There was no resistance recorded for the following combinations: amikacin plus piperacillin-tazobactam, amikacin plus nitrofurantoin and gentamicin plus ni- trofurantoin [Table 5]. R E S U L T S A total of 87 isolates of E. coli and Klebsiella pneumo- niae were isolated from patients admitted to paediat- ric wards in SQUH in 2005. Out of these 13 (14.9 %) were ESBL producers, out of which 6 (46.2%) were E. coli and 7 (53.8%) were Klebsiella pneumoniae [Table 1]. The percentage of E. coli producing ESBL from the total number of E. coli isolated in 2005 was 13.3% [Ta- ble 1]. While the percentage of Klebsiella pneumoniae producing ESBL from the total number of Klebsiella pneumoniae isolated in 2005 was 16.6% [Table 1]. Most of the ESBLs isolates were from urine (46.2%) and blood (42.6%) [Table 2]. A total of 85.7% of ESBL producing Klebsiella pneumoniae were isolated from urine samples, while 83.3% of ESBL producing E. coli were from blood [Table 1]. No ESBL were isolated from wound and pus swabs. The main risk factors for ESBL in these children were the previous exposure to antimicrobials (100%) [Table 3], hospital stays of more than 5 days (92.3%) and female sex (84.6%). Malignancies, admission to the Intensive Care Unit and the use of a urinary cath- eter were each (38.5%) associated with ESBL. Only one patient (1/13) was ventilated. Abdominal surgery and obstructive disease of the urinary tract were not found to be risk factors in our patients. The carbapenems (imipenem and meropenem) were the most active antibiotics against the ESBLs tested, with no resistance recorded [Table 4], followed by amikacin with 8% resistance. All the ESBLs were resistant to oximino-cephalosporins. All ESBL producing Klebsiella pneumoniae were sensitive to gentamycin and amikacin [Table 4], whereas all E. coli were resistant to gentamycin and but only 18% were resistant to amikacin. All Klebsiella pneumoniae were resistant to pipera- cillin-tazobactam and nitrofurantoin, whereas no re- sistance was seen in E. coli to nitrofurantoin and only 16% were resistant to piperacillin-tazobactam. All E. coli were resistant to ciprofloxacin and trimethoprim/ sulfamethoxazole, while 14% of Klebsiella pneumoniae Isolates Total ESBLs ESBL (%) E.coli 45 6 13.3 K. pneumoniae 42 7 16.6 Total 87 13 14.9 Table 1: Percentage of ESBLs among E.coli and K. pneumoniae in Sultan Qaboos University Hospital, Oman, paediatrics wards in 2005 Isolates Blood Respiratory Swabs Urine Klebsiella pnemoniae 1 0 0 6 E. coli 5 1 0 0 Total 6 1 0 6 Table 2: Source of ESBL isolates from paediatric wards Z A K A R I YA A L M U H A R R M I , A K B A R M R A FAY, A B D U L L A H B A L K H A I R , S A L E M A L -TA M E M I , A L I A L M AWA L I A N D H I L A L A L S A D I R I 174 were resistant to ciprofloxacin and 28% were resistant to trimethoprim/sulfamethoxazole [Table 4]. No resistance was recorded for the following com- binations: amikacin plus piperacillin-tazobactam, amikacin plus nitrofurantoin and gentamicin plus ni- trofurantoin [Table 5]. Klebsiella pneumoniae isolates were sensitive to all combinations containing gen- tamicin and amikacin [Table 5]. E. coli isolates were sensitive to all combination containing nitrofurantoin [Table 5]. All patients (100%) were isolated in a single room and nursed using gloves. They were all treated with a carbapenem (imipenem or meropenem). All patients (100%) cleared the infection. D I S C U S S I O N The percentage of ESBL producing E. coli and Kleb- siella pneumoniae in children admitted at SQUH was low compared to other SQUH wards. The prevalence of ESBLs in medical wards was 28.3% (unpublished data); however, the prevalence rate of ESBLs among E. coli and Klebsiella pneumoniae isolated from paedi- atrics patients was significantly high (13.3% and 16.6% respectively) compared to the prevalence of ESBLs in the USA in 2004 (1.4% for E. coli and 4.4% Klebsiella pneumoniae) 31 and Europe (10.8% for E. coli and 13.6% for Klebsiella pneumoniae).31 The rate of ESBL among E. coli (13.3%) was lower than that for Klebsiella pneu- moniae (16.6%). This was the same as the prevalence in USA (1.4% for E. coli versus 4.4% for Klebsiella pneu- moniae) and Europe (10.8% for E. coli versus 13.6% for Klebsiella pneumoniae).31 Urine (70.8%) was the main source of ESBLs from all patients, followed by blood (15%). The high rate of ESBLs in urine samples is not striking if we consider the high prevalence of ESBLs in the gut as shown in Hong Kong where the faecal carriage rates of ESBL is 19% in general outpatients, 19.3% in hospitalized pa- tients, 22.5% in healthy inmates, and 33.3% in conva- lescent patients.35 In one Middle East country, 1.25% of all gram-negative organisms causing community acquired urinary tract infections during 1999 were re- ported as ESBL producers.22, 36 Previous exposure to antimicrobials, female sex, Risk factors Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Total Sex M F F M F F F F F F F F F F=11 M=2 Previous use of antibiotics Y Y Y Y Y Y Y Y Y Y Y Y Y 13/13 Hospital stay > 5 days Y Y Y N Y Y Y Y Y Y Y Y Y 12/13 Malignancies N Y N N N Y Y N Y N N Y N 5/13 Use of urinary catheter N N Y N N N N Y N Y Y N Y 5/13 Admission to Intensive Care Unit Y N N N N Y Y N Y N N Y N 5/13 Ventilatory assistance Y N N N N N N N N N N N N 1/13 Abdominal surgery N N N N N N N N N N N N N 0 Obstructive disease of urinary tract N N N N N N N N N N N N N 0 Table 3: ESBL risk factors in a group of Sultan Qaboos University Hospital paediatric patients E X T E N D E D - S P E C T R U M β - L A C TA M A S E ( E S B L ) I N O M A N I C H I L D R E N 175 prolonged hospital stays of more than 5 days, malig- nancies, admission to the Intensive Care Unit and the use of a urinary catheter were all found to be risk fac- tors for ESBL acquisition. Exposure to antimicrobials was present in 100% of cases, which supports the need for antimicrobial control. 84.6% of cases were female, this might be explained by the increased frequency of urinary tract infections (UTI) in females. All the ESBL isolates in SQUH were susceptible to carbapenems (100%). The susceptibility rate is similar to ESBLs in the USA where they were 100% suscepti- ble to meropenem and imipenem.31 Meropenem and imipenem activity against ESBL producing E. coli and Klebsiella spp. collected in Europe during 1997–2004 was between 96.9–100.0%, which was lower than the susceptibility of ESBLs in SQUH and the USA.31 Sus- ceptibility of ESBLs from SQUH to amikacin was very high (95.9% for E. coli and 90% for Klebsiella pneumo- niae). Kizirgila et al have shown similar susceptibility patterns to amikacin for ESBLs in Turkey (94.5 for E. coli and 83.3% for Klebsiella pneumoniae), 37 which makes amikacin a good antibiotic in treatment of ES- BLs especially in combination therapy. On the contra- ry, gentamicin had very low activity against ESBLs at SQUH. Gentamicin had only (28.8%) activity against E. coli compared to Europe and USA where the E. coli susceptibility to gentamicin was 66.7% and 80% re- spectively in 2004.31 Gentamicin had only 25% activity against Klebsiella pneumoniae which is similar to the USA (26.3%), which are lower rates than those (47.5%) reported in Europe in 2004.31 ESBLs at SQUH had low susceptibility against pip- eracillin/tazobactam (50.7% E. coli and 32.5% Kleb- siella pneumoniae). This level was lower than that re- ported in Europe (72.5% E. coli and 38.6% Klebsiella pneumoniae)31 and the USA (80.0% for E. coli and 42.1% for Klebsiella pneumoniae)31, which does not make piperacillin/tazobactam a good empirical choice if suspicion of ESBL is high. Ciprofloxacin had very low activity against ES- BLs in SQUH. It was only 16.4% active against E. coli which is similar to Europe (20.2%) and the USA (20%) in 2004, 31 whereas higher activity against Klebsiella pneumoniae (32.5%) was recorded. This higher activ- ity compared to E. coli has also been demonstrated in Europe (57.5%) and the USA (36.8%) in 2004.31 The opposite situation has been detected in Turkey where ciprofloxacin was more active against E. coli (33.3%) compared to Klebsiella pneumoniae (25.9%).37 The best non-carbapenem containing combina- tions were amikacin plus piperacillin-tazobactam, amikacin plus nitrofurantoin and gentamycin plus ni- trofurantoin. So if ESBL is expected in a severely ill patient the best empirical combination therapy would be amikacin plus piperacillin-tazobactam. If Klebsiella pneumoniae were cultured and a suspension of ESBL was present, the empirical combination therapy should include either gentamycin or amikacin. If E. coli was isolated from a urine culture of a stable patient nitro- furantoin would be the drug of choice. All patients underwent a good infection control procedure of isolation and barrier nursing according to accepted standards. All patients made a full clinical recovery with microbiologic eradication of ESBLs on carbapenem. Overall prevalence of ESBL-producing isolates in Omani children was high compared to other coun- tries. Prevention and good infection control practices should be our priority because these organisms have very limited treatment options. Modification of risk factors and control of antimicrobials should be con- sidered. Carbapenem should be the drug of choice in treatment of ESBLs, which theoretically may lead to increase in carbapenem-resistant Acinetobacter sp and carbapenem-resistant P. aeruginosa. However, Rob- ert G et al have not seen any increase in carbapenem Isolates CN AK IMI MEM CTX CAZ CEFI CIP TAZ SXT F Klebsiella pnemoniae 0 0 0 0 7 7 7 1 7 2 7 E. coli 6 1 0 0 6 6 6 6 1 6 0 Total 6 (46) 1 (8) 0 0 13 (100) 13 (100) 13 (100) 7 (54) 8 (62) 8 (62) 7 (54) CN = gentamicin, AK = amikacin, IMI = imipenem, MEM = meropenem CTX = cefotaxime, CAZ = ceftazidime, CEFI = cefepime, CIP = ciprofloxacin, TAZ = tazocin, SXT = cotrimoxazole, F = nitrofurantoin. Table 4: ESBL isolates from Paediatric wards: frequency and (%) Z A K A R I YA A L M U H A R R M I , A K B A R M R A FAY, A B D U L L A H B A L K H A I R , S A L E M A L -TA M E M I , A L I A L M AWA L I A N D H I L A L A L S A D I R I 176 resistance despite continued use of meropenem and imipenem.30 Other options would be amikacin plus piperacillin-tazobactam or nitrofurantoin. Wong-Ber- inger suggested the use of piperacillin–tazobactam in the case of a non-outbreak situation, to preserve the therapeutic value of carbapenem.38 C O N C L U S I O N ESBL-producing organisms are becoming a major problem in Omani children. Exposure to antimicro- bials and long admissions are modifiable risk factors that should be targeted for better control. Carbapen- ems are the most sensitive and reliable treatment op- tions for infections caused by ESBLs. Amikacin plus piperacillin-tazobactam or nitrofurantoin are good alternatives. R E F E R E N C E S 1. Winokur PL, Canton R, Casellas JM, Legakis N. 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