Scholarly article on topic 'Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt'

Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt Academic research paper on "Veterinary science"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{"Carbapenem-insensitive Acinetobacter baumannii " / ESBL / "Antimicrobial resistance" / PCR}

Abstract of research paper on Veterinary science, author of scientific article — Mohamed H. Al-Agamy, Noha G. Khalaf, Mahmoud M. Tawfick, Atef M. Shibl, Amany El Kholy

Summary Objectives This study investigated the prevalence of diverse Ambler class β-lactamase-encoding genes in 40 carbapenem-insensitive Acinetobacter baumannii isolates collected from two hospitals in Egypt during the period January–March 2012. Methods The resistance levels to different groups of antimicrobial agents were determined. PCR was used to detect the different Ambler class β-lactamases encoding the following genes: bla TEM, bla SHV, bla CTX-M, bla VEB, bla PER, bla GES, bla VIM, bla IMP , bla SIM , bla SPM , bla GIM, bla NDM, bla ADC, bla OXA-23, bla OXA-24, bla OXA-51, and bla OXA-58. ISAba1 and int1 were detected by PCR. Results The isolates were 100% resistant to amoxicillin–clavulanate, aztreonam, cefepime, cefotaxime, and ceftazidime. Of the isolates, 5% were resistant to colistin, 45% to amikacin, 70% to imipenem, and 85% to ciprofloxacin. The bla ADC- and bla OXA-51-like genes were detected in the entire collection. The prevalences of bla OXA-23, bla OXA-24, and bla OXA-58 were 50%, 7.5%, and 5%, respectively. However, the prevalences of bla TEM-, bla PER-, and bla GES-like genes were 87.5%, 55%, and 27.5%, respectively. SHV, CTX-M, VEB, KPC, and MBL encoding genes were not detected. The ISAba1 was found upstream to bla OXA-51, bla OXA-23, and bla ADC in 85%, 80%, and 50%, respectively. Of note, 45% (18/40) of the isolates co-produced extended-spectrum β-lactamases (PER and GES) and carbapenemases (OXA-23 and OXA-58). Conclusions The bla ADC-, bla TEM-, bla PER-, bla OXA-23-, and bla GES-like genes were found to be the most prevalent types of β-lactamase-encoding gene in A. baumannii collected from Egypt. A high level of carbapenem resistance is mediated by bla OXA-23, bla OXA-24, and bla OXA-58 (minimum inhibitory concentration (MIC) 32 to >256μg/ml), and a low level of carbapenem resistance is mediated by bla GES (MIC 4–16μg/ml) and by up-regulation of ISAba1–OXA-51 (MIC 1–4μg/ml). Class B MBL was not identified to play a role in carbapenem resistance in A. baumannii isolates from Egypt.

Academic research paper on topic "Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt"

Contents lists available at ScienceDirect

International Journal of Infectious Diseases

journal homepage www.elsevier.com/locate/ijid

Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt

Mohamed H. Al-Agamya b *, Noha G. Khalafc, Mahmoud M. Tawfickb, Atef M. Shibla, Amany El Kholyd

a Pharmaceutics and Microbiology Department, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia b Microbiology and Immunology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

c Microbiology and Immunology Department, Faculty of Pharmacy, Modern Arts and Science University, Sixth of October City, Egypt d Clinical Pathology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

CrossMark

ARTICLE INFO

SUMMARY

Article history:

Received 18 October 2013

Received in revised form 1 December 2013

Accepted 2 December 2013

Corresponding Editor: Eskild Petersen,

Aarhus, Denmark

Keywords:

Carbapenem-insensitive Acinetobacter

baumannii

Antimicrobial resistance PCR

Objectives: This study investigated the prevalence of diverse Ambler class p-lactamase-encoding genes in 40 carbapenem-insensitive Acinetobacter baumannii isolates collected from two hospitals in Egypt during the period January-March 2012.

Methods: The resistance levels to different groups of antimicrobial agents were determined. PCR was used to detect the different Ambler class p-lactamases encoding the following genes: blaTEM, blaSHV, biacTx-M, blavEB, blapER, blaGEs, blaViM, blamp, blasiM, blaspM, biaaM, blaNDM, bla^c, biaoxA-23, biaoxA-24, biaOXA-51, and blaOXA-58. ISAbal and intl were detected by PCR.

Results: The isolates were 100% resistant to amoxicillin-clavulanate, aztreonam, cefepime, cefotaxime, and ceftazidime. Of the isolates, 5% were resistant to colistin, 45% to amikacin, 70% to imipenem, and 85% to ciprofloxacin. The blaADC- and blaOXA-51-like genes were detected in the entire collection. The prevalences of blaOxA-23, blaOxA-24, and blaOxA-58 were 50%, 7.5%, and 5%, respectively. However, the prevalences of blaTEM-, blaPER-, and blaGES-like genes were 87.5%, 55%, and 27.5%, respectively. SHV, CTX-M, VEB, KPC, and MBL encoding genes were not detected. The iSAba1 was found upstream to blaOxA-51, blaOXA-23, and blaADC in 85%, 80%, and 50%, respectively. Of note, 45% (18/40) of the isolates co-produced extended-spectrum p-lactamases (PER and GES) and carbapenemases (OXA-23 and OXA-58). Conclusions: The bla^c-, blaTEM-, blaPER-, blaOXA-23-, and blaGES-like genes were found to be the most prevalent types of p-lactamase-encoding gene in A. baumannii collected from Egypt. A high level of carbapenem resistance is mediated by blaOXA-23, blaOXA-24, and blaOXA-58 (minimum inhibitory concentration (MIC) 32 to >256 mg/ml), and a low level of carbapenem resistance is mediated by blaGES (MIC 4-16 mg/ml) and by up-regulation of lSAbaJ-OXA-51 (MIC 1-4 mg/ml). Class B MBLwas not identified to play a role in carbapenem resistance in A. baumannii isolates from Egypt. © 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-

nc-nd/3.0/).

1. Introduction

The genus Acinetobacter comprises Gram-negative, aerobic, glucose-non-fermenting, non-fastidious, non-motile, catalase-positive, and oxidase-negative bacteria. Acinetobacter baumannii is an opportunistic pathogen that is an important source of nosocomial infections, including pneumonia, urinary tract infections, and wound infections, with high mortality.1 In addition, it is often

* Corresponding author. Tel.: +966 553 227 824; fax: +966 1 467 6295. E-mail addresses: malagamy@ksu.edu.sa, elagamy71@yahoo.com (M.H. Al-Agamy).

resistant to a wide variety of antimicrobial agents, including b-lactam antibiotics, fosfomycin, and trimethoprim. Therefore, infections caused by multidrug-resistant A. baumannii are currently among the most difficult to treat.1,2 A variety of molecular mechanisms conferring resistance to b-lactams have been reported in A. baumannii, such as the production of b-lactamases enzymes, alterations in the outer membrane protein, the production of penicillin-binding proteins, and increased activity of efflux pumps.1 However, the most prevalent mechanism of extended-spectrum cephalosporin and carbapenem resistance in A. baumannii is enzymatic degradation by b-lactamases.1-3 The Ambler class A, B, C, and D b-lactamases confer various resistance phenotypes, such as extended-spectrum b-lactamases (ESBLs), metallo-b-lactamases

http://dx.doi.org/10.1016/j.ijid.2013.12.004

1201-9712/© 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

(MBLs), carbapenem-hydrolyzing class D b-lactamases (CHDLs), and Acinetobacter-derived cephalosporinases (ADCs).1,2,4-7 Acquired resistance to carbapenem is mediated most often by the CHDLs (OXA-23, OXA-24/40, OXA-58, and OXA-143) and less frequently by MBLs (VIM, IMP, SPM, GIM, and NDM), which are responsible for high levels of carbapenem resistance.5,7-9 Recently, the Ambler class A carbapenemase GES has been described in A. baumannii, which is responsible for a low level of carbapenem

2 3 10-13

resistance.2,3,;o 13

The resistance of A. baumannii to extended-spectrum cephalosporins is usually related to the over-expression of the resident Ambler class C blaADC gene,6,14,15 or infrequently to the acquisition of ESBL (TEM, SHV, CTX-M, VEB, PER, GES, and KPC) encoding genes.1-4,10-13,16,17 The PER-, VEB-, and GES-like types are the most common Ambler class A b-lactamases in A. baumannii.2-4,10-13,18

There is little information on the frequency of occurrence, prevalence, and distribution of the Ambler class b-lactamases in Egypt. Therefore, this study was undertaken to determine the prevalences of the class A, B, C, and D b-lactamases that confer various b-lactamase resistance phenotypes and to determine the prevalences of ISAbal and class 1 integron among A. baumannii isolates collected from two Egyptian hospitals.

2. Materials and methods

2.1. Bacterial isolates

A total of 40 non-consecutive, unique, imipenem-insusceptible A. baumannii clinical isolates were collected from Kasr El Aini Hospital, Cairo and Dar Al Fouad Hospital, Sixth of October City, Egypt over a period of 3 months from January to March 2012. The isolates were identified using the API 20 NE system (bioMerieux, Marcy l'Etoile, France) and confirmed using PCR to detect the intrinsic blaOXA-51.19

2.2. Determination of the minimum inhibitory concentration (MIC)

The MICs of amikacin, amoxicillin-clavulanate, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, imipenem, gen-tamicin, and colistin were determined for the 40 imipenem-insusceptible A. baumannii isolates using the British Society for Antimicrobial Chemotherapy (BSAC) agar dilution method, with BSAC/European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints.20 Escherichia coli ATCC 25922 was used as the reference strain.

2.3. Phenotypic detection of ESBLs and MBLs

ESBL screening was performed using the disk diffusion method, in accordance with the Clinical and Laboratory Standards Institute (CLSI) recommended guidelines.21 ESBL production was confirmed

with Etest ESBL strips (AB Biodisk, Solna, Sweden). Imipenem/ imipenem + ethylenediaminetetraacetic acid (EDTA) E-test MBL strips (AB Biodisk, Solna, Sweden) were used in accordance with the manufacturer's directions to investigate MBL production. A ratio of the MICs of imipenem to imipenem + EDTA of >8 or the presence of a phantom zone was taken as a positive result.

2.4. Molecular characterization of antimicrobial resistance determinants

A series of PCR reactions were performed to detect the different Ambler class bla genes and mobile genetic elements. Primers were designed to amplify the following bla gene groups: class A, blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaKPC, and blaGESl class B, blaIMP, blaVIM, blaGIM, blaSPM, blaSIM, and blaNDMi class C, blaADCl and class D, blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like.14,22-24 Integrase genes (intl) and IS elements (ISAbal) were amplified by PCR using previously described methods.25,26 PCR mapping experiments using combinations of the ISAbal primers and the OXA-51-like, OXA-23-like, OXA-24-like, OXA-58-like, and ADC-like reverse primers were carried out. All PCR assays were performed using Red Load Taq Master (Jena Bioscience, Jena, Germany) in a Techne thermocycler (Techne, UK). Positive and negative controls were included in all PCR assays.

3. Results

3.1. Antimicrobial resistance pattern

The resistance patterns of the 40 A. baumannii isolates and the MIC distributions of the tested antimicrobial agents are shown in Table 1. A. baumannii isolates were all resistant to amoxicillin-clavulanate, aztreonam, cefepime, ceftazidime, and cefotaxime. The resistance rates to ciprofloxacin, imipenem, and amikacin were 85% (34/40), 70% (28/40), and 45% (18/40), respectively. Colistin showed the highest activity against A. baumannii isolates; the resistance rate was 5% (2/40).

3.2. MICs of tested antibiotics against A. baumannii isolates

The MICs and MIC distributions of amoxicillin-clavulanate, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, amika-cin, ciprofloxacin, and colistin are shown in Tables 1 and 2.

3.3. Prevalence of ESBLs and MBLs

Etest strips for ESBLs were applied to the 40 isolates, with 30 (75%) giving a positive result. The imipenem/imipenem + EDTA Etest gave negative MBL results for all isolates.

Table 1

Minimum inhibitory concentration (MIC) distributions of antimicrobial agents for 40 isolates of Acinetobacter baumannii

Antibiotic Resistance pattern, n (%) Distribution of MIC (mg/ml)

R I S <0.25 0.5 1 2 4 8 16 32 64 128 >256

Amoxicillin-clavulanate 40 (100) 0 0 0 0 0 0 0 0 0 0 0 0 40

Cefotaxime 40 (100) 0 0 0 0 0 0 0 0 0 0 0 0 40

Ceftazidime 40 (100) 0 0 0 0 0 0 0 0 0 0 1 1 38

Cefepime 40 (100) 0 0 0 0 0 0 0 0 0 0 0 3 37

Aztreonam 40 (100) 0 0 0 0 0 0 0 0 0 7 7 4 22

Imipenem 28 (70) 7 (17.5) 5 (12.5) 0 0 2 3 4 3 4 1 3 8 12

Amikacin 18 (45) 9 (22.5) 13 (32.5) 0 0 1 2 3 7 9 4 3 6 5

Ciprofloxacin 34 (85) 0 6(15) 1 2 3 2 1 4 4 2 7 8 6

Colistin 2(5) 0 38 (95) 19 9 8 2 1 1 0 0 0 0 0

R, resistant; I, intermediate; S, susceptible.

Table 2

Clinical data, minimum inhibitory concentrations (MIC), and results of PCR for 40 Acinetobacter baumannii isolates

No. Isolate Clinical data

MIC (mg/l)a

Specimen Hospital Date of collection Gender AMC CT TZ FP AT IP AK CI COL TEM PER GES IS/Aba1 -ADC ISAbal -OXA-51 OXA-23 ISAbal -OXA-23 OXA-24 OXA-58

1 AB1 Wound swab Dar Al Fouad 3/1/2012 Male >256 >256 >256 >256 >256 >256 16 64 0.25 + + - + + + + - - TEM + PER + ADC + OXA-51 + OXA-23 + ISAbal + intl

2 AB3 Blood Dar Al Fouad 6/1/2012 Female >256 >256 >256 >256 >256 256 8 128 0.25 + + - + + + + - -

3 AB5 Wound swab Kasr El Aini 9/1/2012 Male >256 >256 >256 >256 >256 128 16 64 0.25 + + - + + + + - -

4 AB6 Drain Dar Al Fouad 10/1/2012 Male >256 >256 >256 >256 >256 128 16 32 0.5 + + - + + + + - -

5 AB9 ETT Kasr El Aini 15/1/2012 Male >256 >256 >256 >256 >256 >256 16 >256 4 + + - + + + + - -

6 AB13 Wound swab Dar Al Fouad 27/1/2012 Male >256 >256 >256 >256 >256 >256 16 16 0.25 + + - + + + + - -

7 AB18 ETT Kasr El Aini 8/2/2012 Male >256 >256 >256 >256 >256 128 4 128 0.25 + + - + + + + - -

8 AB19 CVP Dar Al Fouad 9/2/2012 Female >256 >256 >256 >256 >256 64 8 64 0.25 + + - + + + + - -

9 AB20 Sputum Kasr El Aini 14/2/2012 Female >256 >256 >256 >256 >256 64 32 256 0.25 + + - + + + + - -

10 AB23 Blood Dar Al Fouad 23/2/2012 Female >256 >256 >256 >256 >256 128 16 32 0.5 + + - + + + + - -

11 AB24 Sputum Kasr El Aini 24/2/2012 Male >256 >256 >256 >256 >256 32 32 >256 1 + + - + + + + - -

12 AB26 ETT Kasr El Aini 27/2/2012 Male >256 >256 >256 >256 >256 256 64 128 0.25 + + - + + + + - -

13 AB28 ETT Kasr El Aini 2/3/2012 Male >256 >256 >256 >256 >256 64 16 128 0.5 + + - + + + + - -

14 AB30 Sputum Kasr El Aini 6/3/2012 Male >256 >256 >256 >256 >256 128 8 64 0.5 + + - + + + + - -

15 AB33 Sputum Kasr El Aini 10/3/2012 Male >256 >256 >256 >256 128 128 32 128 1 + + - + + + + - -

16 AB34 Urine Kasr El Aini 13/3/2012 Female >256 >256 >256 >256 >256 >256 64 >256 1 + + - + + + + - -

17 AB22 Urine Kasr El Aini 18/2/2012 Female >256 >256 >256 >256 >256 >256 128 128 0.5 + - - - + + - - - TEM + ADC + OXA-51 + OXA-23 + ISAbal + intl

18 AB40 Sputum Dar Al Fouad 25/3/2012 Male >256 >256 >256 >256 >256 256 >256 16 8 + - - - + + - - -

19 AB14 Blood Kasr El Aini 31/1/2012 Female >256 >256 >256 >256 128 256 >256 256 1 + - - - + + - - -

20 AB7 Pus Kasr El Aini 11/1/2012 Male >256 >256 >256 >256 >256 >256 >256 256 0.25 + + + + + + TEM + GES + ADC + OXA-51 + OXA-23 + OXA-58 ISAbal + intl

21 AB4 Pus Kasr El Aini 7/1/2012 Male >256 >256 >256 >256 32 128 128 64 0.25 + - - + + - + - TEM + ADC + OXA-51 + OXA-24 + ISAbal + intl

22 AB8 Sputum Kasr El Aini 13/1/2012 Male >256 >256 >256 >256 >256 128 256 128 0.25 + - - + + - + -

23 AB37 Unknown Kasr El Aini 17/3/2012 Female >256 >256 >256 >256 >256 256 128 64 1 + - - + + - + -

24 AB38 Unknown Kasr El Aini 19/3/2012 Male >256 >256 >256 >256 128 >256 8 0.5 1 + + + + TEM + PER + ADC + OXA-51 + OXA-58 + ISAbal + intl

25 AB2 Pus Kasr El Aini 5/1/2012 Female >256 >256 >256 128 64 8 16 1 2 + - + - + - - TEM + GES + ADC + OXA-51 + ISAbal

26 AB10 Unknown Dar Al Fouad 17/1/2012 Female >256 >256 >256 >256 64 16 128 2 0.25 + - + - + - - -

27 AB15 Blood Kasr El Aini 2/2/2012 Male >256 >256 >256 >256 64 16 16 0.25 0.5 + - + - + - - -

28 AB16 Urine Kasr El Aini 5/2/2012 Female >256 >256 >256 >256 64 4 >256 8 0.25 + - + - + - - -

29 AB31 Blood Kasr El Aini 8/3/2012 Male >256 >256 >256 >256 128 4 128 4 1 + - + - + - - -

30 AB32 Pus Kasr El Aini 8/3/2012 Male >256 >256 >256 >256 64 16 8 8 2 + - + - + - - -

31 AB12 Pus Kasr El Aini 23/1/2012 Male >256 >256 >256 >256 >256 8 2 8 0.25 + - + - + - - -

32 AB25 Blood Kasr El Aini 26/2/2012 Male >256 >256 >256 >256 64 8 128 1 0.5 + - + - + - - -

33 AB35 Tissue Kasr El Aini 16/3/2012 Male >256 >256 >256 >256 >256 16 1 128 1 + - + - + - - -

34 AB21 CVP Kasr El Aini 17/2/2013 Female >256 >256 >256 >256 64 4 32 16 0.5 + - + - + - - -

35 AB11 Pus Kasr El Aini 20/1/2012 Male >256 >256 >256 >256 32 2 4 16 0.25 - + - - - - - - ADC + PER + OXA-51 + ISAbal

36 AB17 Urine Dar Al Fouad 6/2/2012 Male >256 >256 64 128 32 4 8 0.5 0.25 - + - - - - - -

37 AB27 Blood Kasr El Aini 1/3/2012 Male >256 >256 >256 >256 32 1 8 64 0.25 - + - - - - - -

38 AB29 Blood Kasr El Aini 5/3/2012 Female >256 >256 >256 >256 32 2 64 8 0.5 - + - - - - - -

39 AB39 Sputum Kasr El Aini 22/3/2012 Female >256 >256 >256 128 32 2 2 1 0.25 - + - - - - - -

40 AB36 Blood Dar Al Fouad 16/3/2012 Male >256 >256 128 >256 32 1 4 2 0.25 + - - - - - - - TEM + ADC + OXA-51 + ISAbal

ETT, Endotracheal tube; CVP, Central venous catheter. a AMC, amoxicillin-clavulanate; CT, cefotaxime; TZ, ceftazidime; FP, cefepime; AT, aztreonam; IP, imipenem; AK, amikacin; CI, ciprofloxacin; COL, Colistin.

All isolates were negative to SHV, CTX-M, VEB, VIM, IMP, SIM, SPM, GIM, and NDM. c ADC, intl, OXA-51, ISAbal were universal.

3.4. Prevalences of the Ambler class p-lactamase-encoding genes

The prevalences of four Ambler class p-lactamase-encoding genes among the 40 A. baumannii isolates are shown in Table 2. The intrinsic p-lactamase gene, blaOXA-51-like, was amplified from all 40 A. baumannii isolates. The blaOXA-23 gene was amplified from 20 isolates (50%). The blaOXA-24/40- and blaOXA-58-like genes were detected in three isolates (7.5%) and two isolates (5%), respectively. Class B MBL genes were not detected in the 40 A. baumannii isolates.

The most prevalent Ambler class A p-lactamase-encoding gene was blaTEM, which was identified in 35 (87.5%) isolates; the next most prevalent gene was blaPER, which was identified in 22 (55%) of the isolates. Ambler class A carbapenemase-encoding gene blaGES was detected in 11 (27.5%) of the 40 A. baumannii isolates. However, blaSHV, blaCTX-M, blaVEB, and blaKPC encoding genes were not detected. The prevalence of blaADC, an Ambler class C cephalosporinase, was 100% in the 40 A. baumannii isolates.

3.5. Upstream regulation of ISAba1

All isolates were found to harbor class 1 integron and iSAba1. The iSAba1 element was found upstream to the corresponding genes blaOXA-51, blaOXA-23, and blaADC in 85% (34/40), 80% (16/20), and 50% (20/40), respectively. However, the iSAba1 element was not found upstream of either blaOXA-24/40 or blaOXA-58 (Table 2).

4. Discussion

Antimicrobial resistance in A. baumannii has become a worldwide problem. The emergence of clinical A. baumannii isolates with diverse antibiotic resistance phenotypes causes difficulties in treating infections caused by this pathogen.3 In the present study, A. baumannii isolates were 100% resistant to amoxicillin-clavulanate, third- and fourth-generation cephalos-porins, and monobactams; however 85% of the isolates were also found to be resistant to ciprofloxacin. Amikacin was found to be an effective drug in the treatment of A. baumannii isolates; 45% of the isolates were resistant. The present study is consistent to some extent with previous studies conducted on A. baumannii collected from Egypt.8,27,28 In the study of Mohamed and Raafat,8 100% of A. baumannii isolates (n = 23) were found to be resistant to the third-and fourth-generation cephalosporins. Furthermore, high resistance rates to amikacin, tobramycin, and ciprofloxacin were found: 100%, 82.6%, and 69.6%, respectively. Ahmed et al.27 reported that A. baumannii isolates (n = 52) were 100% resistant to amoxicillin-clavulanate, ceftazidime, ciprofloxacin, nalidixic acid, and chlor-amphenicol; however, the resistance rates to amikacin, cefepime, and cefradine were 76.9%, 80.8%, and 96.2%, respectively. Nasr and Attalah28 found that all isolates (n = 20) were 100% resistant to ampicillin-sulbactam, ceftazidime, ceftriaxone, ciprofloxacin, and piperacillin-tazobactam. High resistance rates were also observed to amikacin (90%), gentamicin (85%), and doxycycline (75%).

Carbapenems have become the drugs of choice for the treatment of serious nosocomial infections caused by Acineto-bacter; however, carbapenem-resistant strains of A. baumannii have been reported worldwide. In the present study, the majority of the isolates (70%) were resistant to imipenem (MIC >8 mg/ml). Resistance to carbapenems in clinical A. baumannii isolates has been notable recently in Egypt. Few studies have determined the resistance rates for carbapenem in A. baumannii isolates from Egypt. High resistance rates to carbapenems have been observed in Egypt, ranging from 75% to 100% for imipenem and from 61% to 77% for meropenem.8,27-30 The resistance to imipenem reflects a problem that might be described as countrywide. in addition, in the present study, 50% of the isolates displayed unusually high

levels of resistance to imipenem, with MIC values >128 mg/ml. In the Middle East and North Africa, the occurrence of imipenem-resistant A. baumannii is recognized with alarm. The resistance rate of A. baumannii to imipenem was found to be 95% in Turkey, 65% in Saudi Arabia, 47.9% in Algeria, 45% in Tunisia, and 19.14% in Kuwait.12,13,31-33 The emergence of A. baumannii strains with increased carbapenem resistance in this area of the world may be due to the extensive misuse of carbapenems.

Colistin and tigecycline are the last options for the treatment of carbapenem-resistant A. baumannii. Lately, A. baumannii isolates have frequently been found to be resistant to most antimicrobial agents, and evidence of pan-drug resistance among these isolates has been reported. A. baumannii isolates resistant to carbapenems, colistin, and tigecycline have been identified, making the treatment of these isolates particularly difficult. The rate of colistin resistance is relatively low, likely because of its infrequent use. in the present study, colistin retained its activity against most of the tested isolates, with a percentage of susceptibility of 95%. That is consistent with previous studies in Egypt, in which colistin was found to be active against 82.6%8 and 100%30 of the tested isolates. in addition, in other studies, it was found that 100% of A. baumannii was sensitive to colistin in Algeria, 92.5% in Kuwait, and

70.9% in Saudi Arabia.12,31,33

The most prevalent mechanism of carbapenem resistance in A. baumannii is the enzymatic degradation by carbapenem-hydrolyzing p-lactamases. The most widespread carbapenemases in A. baumannii are CHDLs and, to a lesser extent, MBL and class A carbapenemases.1,3,5,12,13,34 MBL, mostly VIM and IMP, has been reported sporadically in some parts of the world.1 MBL NDM-1 and NDM-2 were first described in A. baumannii from Egypt,7,35 and then spread in the Middle East.36 MBL VIM, SPM-1, and G1M-1 were detected previously in A. baumannii isolates from Egypt.8,30 Nevertheless, in the present study, none of the A. baumannii isolates harbored blaiMP, blaViM, blaSPM, blaSiM, blaGiM, or blaNDM MBL-encoding genes.

CHDLs can be divided into four main subgroups: the intrinsic blaOXA-51-like and the acquired carbapenemase genes blaOXA-23-, blaOXA-24/40-, and blaOXA-58-like.1 Numerous studies have recently reported that blaOXA-23 is the most frequent type of carbapenemase identified among carbapenem-resistant A. baumannii.1,12,13,29,30,37 in this study, the most prevalent CHDL-encoding gene in A. baumannii was blaOXA-23, with a prevalence rate of 50% (n = 20), which is in agreement with previous studies.12,13,29 However, Fouad et al.30 detected blaOXA-23 in their entire collection of A. baumannii isolates. blaOXA-24/40 has mostly been found in the Asian and iberian peninsulas, but has also been detected in other areas.1,12,13,38,39 The OXA-58 gene has been reported in isolates of A. baumannii scattered throughout different parts of the world, including Algeria, Argentina, Italy, Kuwait, Turkey, the UK, and the USA.1,37,40-43 In this study, the prevalence of the OXA-58-encoding gene in the clinical isolates of A. baumannii was found to be 5% (2/ 40). In Egypt and Algeria, 9.1% and 14.7%, respectively, of carbapenem-resistant A. baumannii isolates were found to produce OXA-58.29,37 Additionally, in italy and Turkey, carbapenem resistance of A. baumannii has consistently been related to the production of blaOXA-58.41,42 Only a few studies on carbapenemases in A. baumannii in Egypt are available.7,8,29,35 in the present study, all three acquired class D carbapenemases OXA-23-, OXA-24/40-, and OXA-58-encoding genes were identified among the tested strains correlating with resistance to carbapenems, with prevalences of 50%, 7.5%, and 5%, respectively. in a recent study conducted in Egypt by Al-Hassan et al., the prevalences of OXA-23, OXA-40, and OXA-58 were 55.88%, 2.9%, and 14.7%, respectively.29 The prevalence of OXA-23 in the present study is very similar to that found in the study of Al-Hassan et al.; however, the prevalence of OXA-24 in the present study is higher than that found in the

M.H. Al-Agamy et al. /International Journal of Infectious Diseases 22 (2014) 49-54

study of Al-Hassan et al. In addition, we found that the prevalences of blaOXA-23, blaOXA-24, and blaOXA-58 were lower. It is worth noting that the presence of the blaOXA-23 carbapenemase-encoding gene along with the coexistence of blaOXA-58 was detected in one strain in the present study. This finding is in agreement with other

studies.29,37,38

The blaOXA-51-like gene is unique in that it occurs naturally in A. baumannii. Therefore, it is chromosomally located and is widely prevalent. Many studies have indicated that the identification of the blaOXA-51-like gene is a reliable and rapid method to presumptively identify A. baumannii. In addition, the identification of this gene reveals that the rate of antibiotic resistance to various antibiotics is high in A. baumannii isolates.19 Insertion sequences (IS) may contribute to the over-production and dissemination of p-lactamase.25,43 The over-expression of CHDL-encoding genes, driven mostly by promoters provided by their upstream ISs, is one of the means by which A. baumannii acquires a high level of carbapenem resistance. ISAbal and ISAba825 upstream to the blaOXA-51-like gene are associated with the over-expression of the blaOXA-51-like and other CHDL-encoding genes along with carbapenem resistance in A. baumannii.12,13,43 However, some isolates harboring the blaOXA-51-like gene with an upstream ISAbal are still susceptible to carbapenems (Pagano et al.44). The present study revealed that all A. baumannii isolates had blaOXA-51 and ISAba1. The ISAba1 element was found upstream to the corresponding genes blaOXA-51, blaOXA-23, and blaADC in 85% (34/40), 80% (16/20), and 50% (20/40), respectively. However, the ISAbal element was not found upstream of either blaOXA-24/40 or blaOXA-58. The ISAbal element was not found upstream of blaOXA-51 in six (15%) isolates with imipenem MICs ranging from 1 to 4 mg/ml. The up-regulation of ISAbal to blaOXA-51 was found in 34 isolates. Twenty-five out of 34 isolates were concomitant with other CHDLs that had markedly high MICs for imipenem (>32 mg/ml): blaOXA-23 (n = 19), blaOXA-24 (n = 3), blaOXA-58 (n = 1), and one isolate co-produced blaOXA-23 and blaOXA-58. Nine of 34 isolates were not concomitant with other CHDLs, with lower MICs for carbapenems (2-8 mg/ml). In the present study, up-regulation of ISAbal played an important role in the over-expression of blaOXA-51, blaOXA-23, and blaADC.

GES variants and KPC are Ambler class A carbapenemases that have been reported in the last 5 years in A. baumannii;3,12,13,17 our isolates were tested for their encoding genes. Our results revealed that A. baumannii were devoid of KPC, but GES was detected in 27.5% (11/40) of the isolates. In the present study, the prevalence of GES is in agreement with the findings of previous Turkish and Saudi studies,1213 with prevalences of 23.8% and 34.5%, respectively. Several GES-1 mutants have been detected in A. baumannii, such as GES-11, GES-12, GES-14, and GES-22.2,3,10,11,13 Unfortunately, in the current study, GES-encoding genes were not sequenced; however from the MIC data it can be concluded that they may be GES-1 variants, which possess carbapenemase activity. Several studies detecting GES in A. baumannii have been published recently. GES-11 has been reported from Turkey, Egypt, Kuwait, Gaza, and France.3,10,11,13 GES-12 has been detected in Egypt, Belgium, and France, and in addition, GES-14 has been detected in Turkey and Kuwait.3,11 GES-22 has been detected in Turkey.13 GES was detected concomitant with OXA-51 (n = 10) or in combination with OXA-23 plus OXA-58 (n = 1). Ten GES-positive isolates, which had GES plus OXA-51, had imipenem MICs ranging from 4 to 16 mg/ml. This result indicates that the GES-1 mutant is responsible for a high level of carbapenem resistance and/or up-regulation of ISAbal to OXA-51.

Several Ambler class A ESBLs have been identified in A. baumannii, such as CTX-15, PER-1, PER-2, PER-7, and VEB-1.4,16,18 PER and, to a lesser extent, VEB are the most common Ambler class A ESBLs in A. baumannii.4,12,18,45-48 Our isolates were

tested for PER, VEB, TEM, SHV, and CTX-M genes. Our results

revealed that A. baumannii were devoid of VEB, SHV, and CTX-M, but TEM and PER were detected in 87.5% and 49.1% of A. baumannii isolates, respectively. PER has been documented in Acinetobacter isolates from France, Belgium, India, Iran, South Korea, Saudi Arabia, and Argentina.418 45-48 PER-1, PER-2, and PER-7 have been detected previously in A. baumannii.4,45-48 In Iran and Korea, 51% and 54%, respectively, of nosocomial isolates of Acinetobacter spp were found to produce PER-1,18,46 and our results agree with those studies.

The most common mechanism of resistance of A. baumannii to p-lactam antibiotics is attributed to the presence of a chromosomal cephalosporinase-encoding gene.14,15 Most AmpC-type p-lactamases naturally produced by Gram-negative bacteria hydro-lyze amino- and ureidopenicillins, cephamycins, and, at a low level, oxyiminocephalosporins, such as ceftazidime, cefotaxime, ceftri-axone, and aztreonam. Several allelic variants of the A. baumannii AmpC enzyme have also been reported.14 Recently, a uniform designation for this family of cephalosporinases has been suggested: ADC, with AmpCs of A. baumannii.6,14 The enzyme is normally expressed at low levels and is not inducible, but overexpression occurs with the upstream insertion of ISAbal common in A. baumannii, which provides an efficient promoter for the blaAmpC gene. In this study, the entire collection had ADC, 40% of which harbored the upstream ISAbal . The detection of Intl is considered a good indicator for the spread of epidemic Acineto-bacter isolates and it can be responsible for the integration of resistance markers either on the chromosome or plasmid.26 In the present study, the prevalence of Intl was universal; however in a recent study from Egypt, the prevalence of Intl was detected in 85% of Acinetobacter isolates.30

Current knowledge of A. baumannii is presented in this paper. This report highlights the emergence of blaOXA-23-like and blaGES-like genes, especially those conferring carbapenem resistance in A. baumannii. We can conclude that these isolates were devoid of class B MBL. PER-1 is the dominant ESBL and ADC is the dominant extended-spectrum cephalosporinase. ISAbal plays an important role in the over-expression of blaOXA-51. To our knowledge, PER-, GES- and ADC-like have not been reported in A. baumannii from Egypt. Heterogeneous groups of p-lactamases were identified in our isolates.

Acknowledgement

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project (No. RGP-VPP-038).

Conflict of interest: The authors declare that they have no competing interests.

References

1. Abbott I, Cerqueira GM, Bhuiyan S, Peleg AY. Carbapenem resistance in Acine-tobacter baumannii: laboratory challenges, mechanistic insights and therapeutic strategies. Expert Rev Anti Infect Ther 2013;11:395-409.

2. Bonnin RA, Nordmann P, Potron A, Lecuyer H, Zahar JR, Poirel L. Carbapenem-hydrolyzing GES-type extended-spectrum beta-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 2011;55:349-54.

3. Bonnin RA, Rotimi VO, Al Hubail M, Gasiorowski E, Al Sweih N, Nordmann P, et al. Wide dissemination of GES-type carbapenemases in Acinetobacter baumannii isolates in Kuwait. Antimicrob Agents Chemother 2013;57:183-8.

4. Bonnin RA, Potron A, Poirel L, Lecuyer H, Neri R, Nordmann P. PER-7, an extended-spectrum beta-lactamase with increased activity toward broad-spectrum cephalosporins in Acinetobacter baumannii. Antimicrob Agents Che-mother 2011;55:2424-7.

5. Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect 2006;12:826-36.

6. Rodriguez-Martinez JM, Nordmann P, Ronco E, Poirel L. Extended-spectrum cephalosporinase in Acinetobacter baumannii. Antimicrob Agents Chemother 2010;54:3484-8.

7. HrabakJ, Stolbova M, Studentova V, Fridrichova M, Chudackova E, Zemlick-ova H. NDM-1 producing Acinetobacter baumannii isolated from a patient repatriated to the Czech Republic from Egypt. Euro Surveill 2012;17. pii: 20085.

8. Mohamed NM, Raafat D. Phenotypic and genotypic detection of metallo-beta-lactamases in imipenem-resistant Acinetobacter baumannii isolated from a tertiary hospital in Alexandria, Egypt. Res J Microbiol 2011;6:750-60.

9. Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H. OXA-143, a novel carbapenem-hydrolyzing class D ß-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 2009;53:5035-8.

10. Moubareck C, Brémont S, Conroy MC, Courvalin P, Lambert T. GES-11, a novel integron-associated GES variant in Acinetobacter baumannii. Antimicrob Agents Chemother 2009;53:3579-81.

11. Bogaerts P, Naas T, El Garch F, Cuzon G, Deplano A, Delaire T, et al. GES extended-spectrum ß-lactamases in Acinetobacter baumannii isolates in Belgium. Antimicrob Agents Chemother 2010;54:4872-8.

12. Al-Agamy MH, Shibl AM, Ali MS, Khubnani H, Radwan HH, Livermore DM. Distribution of ß-lactamases in carbapenem-non-susceptible Acinetobacter baumannii in Riyadh, Saudi Arabia. J Glob Antimicrob Res 2013. http:// dx.doi.org/10.1016/j.jgar.2013.08.004.

13. Cicek AC, Saral A, Iraz M, Ceylan A, Duzgun AO, Peleg AY, et al. OXA- and GES-type ß-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital. Clin Microbiol Infect 2013 Jul 19. http://dx.doi.org/10.1111/1469-0691.12338.

14. Hujer KM, Hamza NS, Hujer AM, Perez F, Helfand MS, Bethel CR, et al. Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 ß-lactamase: defining a unique family of class C enzymes. Antimicrob Agents Chemother 2005;49:2941-8.

15. Villalon P, Valdezate S, Medina-Pascual MJ, Carrasco G, Vindel A, Saez-Nieto JA. Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-ß-lactamase genes, and of common insertion sequences, in epidemic clones of Acinetobacter baumannii from Spain. J Antimicrob Chemother 2013;68:550-3.

16. Potron A, Munoz-Price LS, Nordmann P, Cleary T, Poirel L. Genetic features of CTX-M-15-producing Acinetobacter baumannii from Haiti. Antimicrob Agents Chemother 2011;55:5946-8.

17. Robledo IE, Aquino EE, Vazquez GJ. Detection of the KPC gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii during a PCR-based nosocomial surveillance study in Puerto Rico. Antimicrob Agents Chemother 2011;55:2968-70.

18. Farajnia S, Azhari F, Alikhani MY, Hosseini MK, Amir P, Sohrabi N. Prevalence of PER and VEB type extended spectrum beta lactamases among multidrug resistant Acinetobacter baumannii isolates in north-west of Iran. Iran J Basic MedSci 2013;16:751-5.

19. Akbari M, Niakan M, Taherikalani M, Feizabadi MM, Azadi NA, Soroush S, et al. Rapid identification of Iranian Acinetobacter baumannii strains by single PCR assay using blaOXA-51-like carbapenemase and evaluation of the antimicrobial resistance profiles of the isolates. Acta Microbiol Immunol Hung 2010;57:87-94.

20. Howe RA, Andrews JM. BSAC standardized disc susceptibility testing method (version 11). BSAC Working Party on Susceptibility Testing. J Antimicrob Chemother 2012;67:2783-4.

21. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: 19th informational supplement. CLSI document M100-S19. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

22. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother 2010;65:490-5.

23. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119-23.

24. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acineto-bacter spp. Int J Antimicrob Agents 2006;27:351-3.

25. Segal H, Garny S, Elisha BG. Is ISAba-1 customized for Acinetobacter? FEMS Microbiol Lett 2005;243:425-9.

26. Koeleman JG, Stoof J, van der Bijl MW, Vandenbroucke-Grauls CM, Savelkoul PH. Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J Clin Microbiol 2001;39:8-13.

27. Ahmed SH, Abdelwahab SF, Hasanen AM, Mohammed DS. Multidrug resistant Egyptian isolates of Acinetobacter baumannii. J Am Sci 2011;7:1013-9.

28. Nasr RA, Attalah MF. Molecular epidemiology of nosocomial Acinetobacter baumannii isolates. Nature and Science 2012;10:76-82.

29. Al-Hassan L, El Mehallawy H, Amyes SG. Diversity in Acinetobacter baumannii isolates from paediatric cancer patients in Egypt. Clin Microbiol Infect 2013 Feb 15. http://dx.doi.org/10.1111/1469-0691.12143.

30. Fouad M, Attia AS, Tawakkol WM, Hashem AM. Emergence of carbapenem-resistant Acinetobacter baumannii harboring the OXA-23 carbapenemase in intensive care units of Egyptian hospitals. Int J Infect Dis 2013;17:e1252-4. http://dx.doi.org/10.1016/j.ijid.2013.07.012.

31. Bakour S, Touati A, Sahli F, Ameur AA, Haouchine D, Rolain JM. Antibiotic resistance determinants of multidrug-resistant Acinetobacter baumannii clinical isolates in Algeria. Diagn Microbiol Infect Dis 2013;76:529-31.

32. Ben Othman A, Zribi M, Masmoudi A, Abdellatif S, Ben Lakhal S, Fendri C. Phenotypic and molecular epidemiology of Acinetobacter baumannii strains isolated in Rabta Hospital, Tunisia. Arch Inst Pasteur Tunis 2007;84:11-9.

33. Al-Sweih NA, Al-Hubail M, Rotimi VO. Three distinct clones of carbapenem-resistant Acinetobacter baumannii with high diversity of carbapenemases isolated from patients in two hospitals in Kuwait. J Infect Public Health 2012;5:102-8.

34. Nordmann P. Gram-negative bacteria with resistance to carbapenems. Med Sci (Paris) 2010;26:950-9.

35. Kaase M, Nordmann P, Wichelhaus TA, Gatermann SG, Bonnin RA, Poirel L. NDM-2 carbapenemase in Acinetobacter baumannii from Egypt. J Antimicrob Chemother 2011;66:1260-2.

36. Espinal P, Poirel L, Carmeli Y, Kaase M, Pal T, Nordmann P, et al. Spread of NDM-2-producing Acinetobacter baumannii in the Middle East. J Antimicrob Chemother 2013;68:1928-30.

37. Touati M, Diene SM, Racherache A, Dekhil M, Djahoudi A, Rolain JM. Emergence of blaOXA-23 and blaOXA-58 carbapenemase-encoding genes in multidrug-resistant Acinetobacter baumannii isolates from University Hospital of Annaba, Algeria. Int J Antimicrob Agents 2012;40:89-91.

38. Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 2010;65:233-8.

39. Acosta J, Merino M, Viedma E, Poza M, Sanz F, Otero JR, et al. Multidrug-resistant Acinetobacter baumannii harboring OXA-24 carbapenemase, Spain. Emerg Infect Dis 2011;17:1064-7.

40. Coelho J, Woodford N, Afzal-Shah M, Livermore D. Occurrence of OXA-58-like carbapenemases in Acinetobacter spp. collected over 10 years in three continents. Antimicrob Agents Chemother 2006;50:756-8.

41. Metan G, Sariguzel F, Sumerkan B, Reijden TV, Dijkshoorn L. Clonal diversity and high prevalence of OXA-58 among Acinetobacter baumannii isolates from blood cultures in a tertiary care centre in Turkey. Infect Genet Evol 2013;14:92-7.

42. Migliavacca R, Espinal P, Principe L, Drago M, Fugazza G, Roca I, et al. Characterization of resistance mechanisms and genetic relatedness of carbapenem-resistant Acinetobacter baumannii isolated from blood, Italy. Diagn Microbiol Infect Dis 2013;75:180-6.

43. Lopes BS, Al-Hassan L, Amyes SG. lSAba825 controls the expression of the chromosomal blaOXA-51-like and the plasmid borne blaOXA-58 gene in clinical isolates of Acinetobacter baumannii isolated from the USA. Clin Microbiol Infect 2012;18:E446-51.

44. Pagano M, Martins AF, Machado AB, Barin J, Barth AL. Carbapenem-susceptible Acinetobacter baumannii carrying the ISAbal upstream blaOXA-51-like gene in Porto Alegre, southern Brazil. Epidemiol Infect 2012;19:1-4.

45. Pasteran F, Rapoport M, Petroni A, Faccone D, Corso A, Galas M, et al. Emergence of PER-2 and VEB-1a in Acinetobacter baumannii strains in the Americas. Antimicrob Agents Chemother 2006;50:3222-4.

46. Yong D, Shin JH, Kim S, Lim Y, Yum JH, Lee K, et al. High prevalence of PER-1 extended-spectrum beta-lactamase-producing Acinetobacter spp. in Korea. Antimicrob Agents Chemother 2003;47:1749-51.

47. Litake GM, Ghole VS, Niphadkar KB, Joshi SG. PER-1-type extended-spectrum beta-lactamase-producing Acinetobacter baumannii clinical isolates from lndia. Int J Antimicrob Agents 2009;34:388-9.

48. Naas T, Bogaerts P, Bauraing C, Degheldre Y, Glupczynski Y, Nordmann P. Emergence of PER and VEB extended-spectrum beta-lactamases in Acinetobacter baumannii in Belgium. J Antimicrob Chemother 2006;58:178-82.