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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1030-1036

Phenotypic and molecular characterization of clinical Acinetobacter isolates from Menoufia University Hospitals


Department of Microbiology and Immunology, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt

Date of Submission18-Jun-2017
Date of Acceptance13-Aug-2017
Date of Web Publication04-Apr-2018

Correspondence Address:
Soma E Ajlan
Shebin Elkom City, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_452_17

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  Abstract 


Objectives
The aims of this study were to investigate the prevalence of Acinetobacter spp. in Menoufia University Hospitals, to investigate their antimicrobial susceptibility patterns, and to assess carbapenemases production in these isolates.
Background
Acinetobacter spp. are important opportunistic pathogens responsible for nosocomial infections.
Materials and methods
This study was conducted on 603 clinical samples from patients admitted to Menoufia University Hospitals. Acinetobacter spp. were identified by standard microbiological methods and API20NE test kits. Antimicrobial susceptibility was tested using disk diffusion and agar dilution methods. Imipenem-resistant Acinetobacter isolates were further tested for metallo-β-lactamase (MβL) production.
Results
This study was conducted at Medical Microbiology and Immunology Laboratory, Faculty of Medicine, Menoufia University. Acinetobacter spp. represented 10.6% of all collected nosocomial isolates. With regards to API20NE results, A. baumanii was the predominant Acinetobacter spp. (80.8%) followed by A. baumanniiA. calcoaceticus complex (7.7%), A. lwoffii (5.8%), A. haemolyticus (3.8%), and A. pitti (2.6%). Acinetobacter isolates were highly resistant to cefepime (92.3%), ampicillin–sulbactam, piperacillin, piperacillin–tazobactam, ceftazidime, tobramycin (91% for each), amikacin (84.6%), and imipenem (67.9%). Overall, 56.4% of Acinetobacter isolates were susceptible to tigecycline. On agar dilution method, 96.2% of Acinetobacter isolates were found to be susceptible to colistin and 66.7% were imipenem resistant. Imipenem/ethylenediaminetetraacetic acid combined disk test showed that 81.1% of imipenem-resistant Acinetobacter were MβL producers, and multiplex PCR showed that 15.1% of imipenem-resistant Acinetobacter were positive for blaVIM2, but none of them were positive for blaIMP1 gene.
Conclusion
Acinetobacter spp. are serious nosocomial pathogens with high prevalence of carbapenems resistance. Production of carbapenemases, especially MβLs, is considered the main carbapenem-resistance mechanism. Tigecycline and colistin can be valuable therapeutic options for the treatment of Acinetobacter infections.

Keywords: Acinetobacter, β-lactamases, carbapenems, colistin, resistance


How to cite this article:
Baker AM, Makled AF, Salem EH, Salama AA, Ajlan SE. Phenotypic and molecular characterization of clinical Acinetobacter isolates from Menoufia University Hospitals. Menoufia Med J 2017;30:1030-6

How to cite this URL:
Baker AM, Makled AF, Salem EH, Salama AA, Ajlan SE. Phenotypic and molecular characterization of clinical Acinetobacter isolates from Menoufia University Hospitals. Menoufia Med J [serial online] 2017 [cited 2018 May 21];30:1030-6. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1030/229225




  Introduction Top


Acinetobacter spp. are challenging pathogens responsible for serious nosocomial infections such as nosocomial and ventilator-associated pneumonia, urinary tract infections, septicemia, wound sepsis, meningitis, and endocarditis[1]. They can survive in the clinical environment with great resistance to disinfectants, antibiotics, moist, and dry conditions, as they can use different metabolic sources and have the ability to form biofilms[2].

Nosocomial infections by Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) group represent a problem in healthcare settings, with high rates of antibiotic resistance. This group of organisms is responsible for up to 40% of ICU infections[2].

The extensive use of antimicrobial chemotherapy, particularly carbapenems, has contributed to the emergence of carbapenem-resistant Acinetobacter, which usually exhibits a multidrug-resistant phenotype[3]. Carbapenem resistance may be because of several mechanisms including production of β-lactamases especially metallo-β-lactamases (MβLs), outer membrane impermeability, increased expression of efflux pumps, and penicillin-binding proteins modification[1].

Acinetobacter spp. acquires carbapenem resistance by producing MβLs that belong to Ambler class B β-lactamases based on their amino acid sequence homology and to group 3 according to the Bush classification based on their substrate profiles (imipenem hydrolysis). These enzymes require a zinc ion for their activity, which is inhibited by metal chelates, such as ethylenediaminetetraacetic acid (EDTA)[4]. Six groups of MβLs have been described including imipenemase type metallo-β-lactamase (IMP), Verona imipenemase (VIM), Seoul imipenemase (SIM), German imipenemase (GIM), New Delhi metallo-β-lactamase (NDM), and Sao Paulo imipenemase (SPM) carbapenemases[5].

Since the first descriptions of MβLs in Acinetobacter spp., IMP and VIM types have been found to have a wide geographical occurrence[6]. VIM enzymes have been grouped into three main clusters designated as VIM1, VIM2, and VIM7, where VIM1 is normally restricted to Enterobacteriaceae isolates and VIM2 is more widespread among P. aeruginosa isolates. The IMP gene has at least 27 unique variants and are mostly found in Enterobacteriaceae, A. baumannii, and P. aeruginosa isolates[7].

The aims of our study were to investigate the prevalence of Acinetobacter spp. among different clinical samples collected from Menoufia University Hospitals, to determine their antibiotic susceptibility patterns, and to detect carbapenemases production by phenotypic and genotypic methods.


  Materials and Methods Top


This study was conducted at Medical Microbiology and Immunology Department, Faculty of Medicine, Menoufia University. A total of 603 clinical samples were collected from hospitalized patients aged from 5 days to 69 years. The study protocol was approved by the Local Ethics Committee of Menoufia University. Informed consent was obtained from the patients before beginning of the study. All the selected patients were subjected to the following: (i) personal history including name, age, sex, occupation, socioeconomic status, and residence; (ii) clinical history including the duration and the cause of hospital admission (wards or ICUs), exposure to invasive procedure (surgery, urinary catheters, central venous lines, or endotracheal tubes), and if there are any associated comorbidities (diabetes, hypertension, obesity, chronic lung diseases, chronic renal diseases, chronic liver diseases, and immunosuppression).

Clinical sampling

  1. Respiratory secretions were collected in a screw-capped, sterile, wide-mouthed plastic container from morning sputum samples, endotracheal aspirates (suctioning of 1–10 ml of purulent secretions from the endotracheal tube), and bronchial aspirates (during bronchoscopy)
  2. Blood samples were collected by withdrawing 10 ml of venous blood from each adult patient and 2–5 ml from infants and children under strict aseptic conditions and inoculated into culture bottles
  3. For urine samples, 10–20 ml of mid-stream urine was collected from uncatheterized patients in a sterile, dry, wide-neck, leak-proof container. For catheterized patients, samples were collected after disinfection of the collection part of the catheter by discarding the first few drops of urine and collecting 5–10 ml urine using a sterile syringe
  4. Infected wounds were examined through aspiration of pus using a sterile syringe and samples were collected using a sterile cotton swab rolled in all directions
  5. Ascetic fluid samples were aspirated using a sterile syringe
  6. Pleural fluid samples of about 20–40 ml were obtained using a syringe under aseptic conditions. Specimens were collected in tubes containing anticoagulants to avoid clotting and cell clumping
  7. Cerebrospinal fluid (CSF) samples were collected by lumber puncture; three or more separate tubes of CSF are collected[8].


Bacterial isolates

Clinical samples (117 sputum, 45 tracheal aspirates, 24 bronchial aspirates, 66 pus, 126 urine, 114 blood, 66 burn swabs, 15 pleural aspirates, 18 ascetic fluid, and 12 CSF) were collected, processed, and cultured on different media (Oxoid, Basingstoke, UK), namely, nutrient agar, blood agar, Mannitol salt agar, MacConkey agar, and Sabouraud dextrose agar. Patients already on antibiotic were excluded. The growing Acinetobacter isolates were identified by colonial morphology, Gram staining, standard biochemical tests, and API20NE test kits[8]. Confirmed Acinetobacter isolates were preserved on tryptic soy broth with 15% glycerol and frozen at −80°C[9].

Antimicrobial susceptibility

Antimicrobial susceptibility testing for Acinetobacter isolates was performed using disk diffusion method against different antimicrobial agents (Oxoid) as recommended by Clinical Laboratory Standard Institute (CLSI) including the following: piperacillin (100 μg), ampicillin/sulbactam (10/10 μg), piperacillin/tazobactam (100/10 μg), ceftazidim (30 μg), cefepime (30 μg), meropenem (10 μg), imipenem (10 μg), amikacin (30 μg), tobramycin (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), trimethoprim–sulfamethoxazole (1.25/23.75 μg), and tigecycline (30 μg). Minimal inhibitory concentration of imipenem and colistin (Sigma Aldrich, St Louis, Missouri, USA) was determined using agar dilution method according to CLSI guidelines[10].

Metallo-β

-lactamase detection by phenotypic method


Imipenem-resistant Acinetobacter isolates were further tested for MβLs production by imipenem/EDTA (Sigma Aldrich) combined disk test (as the increase in the inhibition zone size for more than 7 mm in the EDTA-containing imipenem disk compared with imipenem disk was considered positive[11]) [Figure 1].
Figure 1: Detection of class B carbapenemase production among Acinetobacter isolates using combined imipenem/ethylenediaminetetraacetic acid (EDTA) synergy test. Letter A represents imipenem disk alone and letter B represents imipenem/EDTA combined disk. There was an expansion of the bacterial growth inhibition zones around combined disc by 7 mm or more in diameter.

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Molecular detection of blaIMP1 and blaVIM2

Bacterial DNA was extracted and purified using the Quick-g DNA Mini Prep Kit (Zymo Research, Irvine, California, USA). Primers sequences (blaVIM2 F: GATGGTGTTTGGTCGCATA, blaVIM2 R: CGAATGCGCAGCACCAG, blaIMP1 F: GGAATAGAGTGGCTTAAYTCTC, and blaIMP1 R: GGTTTAAYAAAACAACCACC[9]) were shipped and received in a lyophilized state (Invitrogen by Thermo Fisher Scientific, Waltham, Massachusetts, USA). The volume of molecular grade H2O added to the lyophilized primer was determined by reading the number of nmol of primers in the tube and multiplied by 10 to make a 100 μmol/l primer stock. Each PCR mixture (25 μl) consisted of 12.5 μl master mix, 10 μl of the extracted DNA sample, 0.5 μl of each primer, and 0.5 μl molecular grade H2O. Multiplex PCR program was performed in a thermal cycler (Biometra, Gottingen, Germany) that consisted of an initial denaturation step at 94°C for 3 min, followed by repeated 35 cycles of DNA denaturation at 94°C for 45 s, primer annealing at 57°C for 45 s, and primer extension at 72°C for 1 min. Finally, an extended 72°C step for 5 min was recorded to ensure that all of the products are of full length[9]. The amplified DNA was electrophoresed (Biometra) using 1.5% agarose gel (Bioline, London, UK) stained with ethidium bromide (Sigma Aldrich), and the bands [Figure 2] were visualized under ultraviolet transilluminator (Biometra) and photographed (Samsung, Seoul, South Korea)[12].
Figure 2: Agarose gel electrophoresis for the multiplex PCR amplified products of Acinetobacter isolates for blaVIM2 and blaIMP1 genes. Lane M, DNA molecular size marker (100–1000 bp); lanes 3, 4, and 6, positive blaVIM2 (390 bp) and negative blaIMP1 (232 bp); lanes 2, 5, 7, 8, and 9, negative samples for both blaIMP1 and blaVIM2.

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Statistical analysis

The data collected were tabulated and analyzed using statistical package for the social sciences (SPSS, version 20; SPSS Inc., Chicago, Illinois, USA) software, on an IBM compatible computer. The results were expressed as number and percentage. χ2-Test was performed (P< 0.05 and <0.001 were considered significant and highly significant, respectively), and accuracy was represented using the terms sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy.


  Results Top


This study was conducted at Medical Microbiology and Immunology Department, Faculty of Medicine, Menoufia University. During this study, 733 pathogens were isolated from 603 different clinical samples. A total of 78 (10.6%) Acinetobacter spp. were identified. Acinetobacte r spp. were derived mostly from respiratory samples (47.4%) followed by blood samples (32.1%), pus swabs (9%), burn swabs (7.7%), and urine samples (3.8%) [Table 1]. Regarding API20NE results, A. baumanii was the predominant Acinetobacter spp. (80.8%) followed by A. baumannii–A. calcoaceticus complex (7.7%), A. lwoffii (5.8%), A. haemolyticus (3.8%), and A. pitti (2.6%).
Table 1: Distribution of clinical Acinetobacter isolates according to different samples among hospital departments

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By disk diffusion method, 92.3% of Acinetobacter isolates were found to be resistant to cefepime, 91% were resistant to ampicillin–sulbactam, 88.5% were resistant to piperacillin, piperacillin–tazobactam, ceftazidime, and tobramycin, 84.6% were resistant to amikacin, 79.5% were resistant to tetracyclines and ciprofloxacin, 76.9% were resistant to trimethoprim–sulfamethoxazole, and 74.4% were resistant to levofloxacin. On the contrary, 56.4 and 78.2% of Acinetobacter isolates were susceptible to tigecycline and colistin, respectively. Moreover, the resistance rates of Acinetobacter isolates for imipenem and meropenem were 67.9 and 64.1%, respectively [Table 2]. Using agar dilution method, 96.2% of Acinetobacter isolates were found to be susceptible to colistin and 66.7% of Acinetobacter isolates were imipenem resistant [Table 3].
Table 2: Antimicrobial susceptibility pattern of Acinetobacter isolates by agar dilution method

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Table 3: Acinetobacter imipenem and colistin susceptibility testing by disk diffusion and agar dilution methods

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On combined disk test, 42/53 (81.1%) of imipenem nonsusceptible Acinetobacter isolates were MβLs producers. On multiplex PCR, 8/53 (15.1%) of imipenem-resistant Acinetobacter isolates were positive for blaVIM2, but none of them was positive forblaIMP1 gene [Table 4].
Table 4: Sensitivity and specificity of combined imipenem/ethylenediaminetetraacetic acid synergy test in relation to polymerase chain reaction for detection of blaVIM2 among Acinetobacter isolates

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  Discussion Top


This study was carried out in Medical Microbiology and Immunology Department, Faculty of Medicine, Menoufia University. Overall, 733 pathogens were isolated from 603 clinical samples that were collected from patients admitted to different departments and ICUs of Menoufia University Hospitals. Of them, 78 (10.6%) were identified as Acinetobacter spp. Similar results were obtained by Fouad et al.[13] in Cairo, who found that Acinetobacter spp. represented 10% of the total isolates. Despite the increasing prevalence of Acinetobacter infection, many clinicians and microbiologists still lack appreciation of the importance of these organisms in the hospitals[13].

In this study, most of Acinetobacter spp. were isolated from ICUs (52.6%) and from respiratory samples (47.4%). In agreement, Al-Saleem[14] in Baghdad isolated 43.48% of Acinetobacter spp. from ICUs and Fouad et al.[13]in Cairo isolated45% of Acinetobacte r isolates from respiratory samples. In accordance with these findings, the respiratory tract is the main site of Acinetobacter infection.

Regarding antimicrobial susceptibility by disk diffusion method, our study revealed high rates of resistance to ampicillin–sulbactam, cefepime (91% for each), piperacillin, ceftazidime (88.5% for each), piperacillin–tazobactam (84.6%), tobramycin (80.8%), amikacin, and tetracyclines (75.6% for each). This was matched with the results of Fouad et al.[13]in Cairo who found high resistance rate of Acinetobacter spp. to antibiotics: 100% for each of ceftazidime, cefepime, ciprofloxacin, and piperacillin/tazobactam and 80% for amikacin, aztreonam, gentamicin, and tobramycin. On the contrary, 56.4% of Acinetobacter spp. were susceptible to tigecycline in the present study. Behera et al.[15] and Koripella et al.[16] in India showed that 42 and 91.3% of isolated Acinetobacter, respectively, were tigecycline sensitive.

Regarding colistin susceptibility, 61/78 (78.2%) and 75/78 (96.25%) of Acinetobacter isolates in this study were colistin susceptible on disk diffusion and agar dilution method, respectively, with high statistically significant difference between the two methods (P > 0.001). This result was in agreement with the CLSI recommendations that depended only on minimal inhibitory concentration interpretations in detection of colistin susceptibility, considering colistin agar dilution method gives unreliable results[10]. The high susceptibility rate of Acinetobacter isolates to colistin (96.2%) by agar dilution method in this study was in agreement with Fouad et al.[13] in Cairoand Fattouh and Nasr El-Din[12] in Sohag who reported that all isolated Acinetobacter spp. (100%) were susceptible to colistin. So, colistin and tigecycline can be the last treatment options left for the management of Acinetobacter infections[12].

Regarding carbapenem susceptibility, 53/78 (67.9%) and 52/78 (66.7%) of Acinetobacter isolates in the current study were imipenem resistant on disk diffusion and agar dilution methods, respectively, with no statistically significant difference between the two methods (P > 0.05). This was matched by Fattouh and Nasr El-Din[12] in Sohag who reported that 71.4% of Acinetobacter isolates were imipenem resistant. In disagreement to our study, Daef et al.[17] in Assiut and Koripella et al.[16] in India reported that only 31.4 and 15.6% of Acinetobacter isolates were imipenem resistant, respectively. However, the study conducted in Saudi Arabia by Mohamed and Al-Ahmady[7] revealed that 95% of isolated Acinetobacter spp. were imipenem resistant.

Imipenem disk screen test was recommended by the Dutch Working Party on the Detection of Highly Resistant Microorganisms for detection of all different class A carbapenemases and VIM MβLs[18].

Carbapenems are the main drugs used in the treatment of Acinetobacter infections. The extensive unwise usage may have led to selection of resistant strains. Hence, the high rate of resistance developed against them, with the main mechanism being production of carbapenemases, especially enzymes belonging to classes B (MβLs)[12]. The present study revealed that 42/53 (79.2%) of imipenem-resistant Acinetobacter isolates were MβLs producers phenotypically by imipenem/EDTA combined disk. Moreover, Zarrilli et al.[19] in Italy observed that 86.7% of imipenem nonsusceptible Acinetobacter isolates were MβLs producers.

Regarding multiplex PCR results, blaIMP1 gene was completely absent, whereas blaVIM2 was detected in only 15.1% of our isolates. Similar results were observed in Saudi Arabia by Mohamed and Al-Ahmady[7] and in Sohag by Fattouh and Nasr El-Din[12], where blaIMP1 was not detected in any of their isolates. Alm El-Din et al.[20] in Tanta detected blaVIM gene in 15.38% of their Acinetobacter isolates. Comparable results were obtained by Fattouh and Nasr El-Din[12] who could not detect blaVIM in any of their isolates and by Mohamed and Al-Ahmady[7] who detected blaVIM in 85% of their Acinetobacter isolates. They reported that the presence of blaIMP1 and blaVIM2 in only eight isolates from 42 tested MBL-positive Acinetobacter strains in this study could be because of the presence of unidentified MBL genes, the limitation of the primer set used, as there are many variants of IMP/VIM gene and the presence of MBL genes other than IMP/VIM[7].

The sensitivity and specificity of imipenem/EDTA combined disk test were 100 and 24.4%, respectively, in this study. All metallo-carbapenemase PCR positive isolates (eight isolates for blaVIM2) were positive in imipenem/EDTA combined disk test. The same observations were obtained by Valenza et al.[21] in Germany who investigated 489 imipenem nonsusceptible clinical isolates and found that, in relation to PCR results, imipenem/EDTA combined disk test was able to discriminate between all MβL-positive and MβL-negative isolates and reported that an initial imipenem susceptibility screening followed by a confirmatory imipenem/EDTA combined disk test represents a valid and less expensive alternative to the molecular investigation of MβL genes and so MβLs detection could be possible not only in reference laboratories but also in routine diagnostic microbiology laboratories[21].


  Conclusion Top


Acinetobacter infection is a common threat in hospital acquired infections (10.6% prevalence rate) particularly in ICUs (52.6%). There is an increase of antibiotic-resistance patterns, especially against carbapenems (67.9%). Carbapenem-resistance patterns among Acinetobacter in this study are alarming. The main mechanism of resistance being the production of carbapenemases, especially MβLs (81.1%). Imipenem/EDTA combined disk test could be a valid alternative to the molecular investigation of MβL genes. Colistin and tigecycline can be the last treatment options left for the management of Acinetobacter infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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