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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 85-89
 

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry rapid detection of carbapenamase activity in Acinetobacter baumannii isolates


1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Chemical Pathology, High Institute of Research, Alexandria University, Alexandria, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Web Publication16-Mar-2017

Correspondence Address:
Noha Abouseada
Department of Medical Microbiology and Immunology, Faculty of Medicine, Alexandria University, Alkhartoum Square, Azarita, Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.202335

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 ~ Abstract 


Introduction: Carbapenamase-producing Acinetobacter baumannii are an increasing threat in hospitals and Intensive Care Units. Accurate and rapid detection of carbapenamase producers has a great impact on patient improvement and aids in implementation of infection control measures. Aim: In this study, we describe the use of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF MS) to identify carbapenamase-producing A. baumannii isolates in up to 3 h. Isolates and Methods: A total of 50 A. baumannii isolates (of which 39 were carabapenamase producers) were tested using MALDI TOF MS. Isolates were incubated for 3 h with 0.25 mg/ml up to 2 mg/ml of imipenem (IMP) at 37°C. Supernatants were analysed by MALDI TOF to analyse peaks corresponding to IMP (300 Da) and an IMP metabolite (254 Da) using UltrafleXtreme (Bruker Daltonics, Bremen, Germany). Results: All carbapenamase-producing isolates were evidenced by the disappearance or reduction in intensity of the 300 Da peak of IPM and the appearance of a 254 Da peak of the IPM metabolite. In isolates that did not produce carbapenamase, the IPM 300 Da peak remained intact. Conclusion: MALDI TOF is a promising tool in the field of diagnostic microbiology that has the ability to transfer identification and antimicrobial susceptibility testing time from days to hours.


Keywords: Acinetobacter, carbapenamases, matrix-assisted laser desorption ionisation time of flight


How to cite this article:
Abouseada N, Raouf M, El-Attar E, Moez P. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry rapid detection of carbapenamase activity in Acinetobacter baumannii isolates. Indian J Med Microbiol 2017;35:85-9

How to cite this URL:
Abouseada N, Raouf M, El-Attar E, Moez P. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry rapid detection of carbapenamase activity in Acinetobacter baumannii isolates. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 25];35:85-9. Available from: http://www.ijmm.org/text.asp?2017/35/1/85/202335





 ~ Introduction Top


Acinetobacter baumannii is recognised as a major bacterial-resistant pathogen.[1] Resistance may extend to include all available therapeutic agents, for example, β-lactams, fiuoroquinolones and aminoglycosides, exerting a great challenge to antimicrobial therapy and infection control programmes.[1],[2]A. baumannii is increasingly shown to be resistant to carbapenems, which are considered powerful β-lactam antimicrobials used for the treatment of nosocomial and community-acquired Gram-negative bacilli, due to presence of the class B metallo-β-lactamases (MBLs) and by class A and D serine carbapenemases.[3],[4] The dissemination of these enzymes among A. baumannii is a global threat.[5] Screening of carbapenemases is, therefore, a cornerstone for appropriate treatment and for the implementation of infection control programmes.[6]

Phenotypic tests such as carbapenamase/carbapenemase inhibitors combination disc test and Hodge test are available, but false results are detected and also are time consuming (18–24 h).[7],[8] Molecular identification of carbapenemases is also available, but they are both money consuming and need experience.[9] Matrix-assisted laser desorption ionisation time of fiight (MALDI TOF) mass spectrometry (MS) has been introduced in microbiology laboratories for routine identification of bacteria and fungi for almost a decade, and now it has been proved to be comparable to sequencing methods for identification.[10] It is accurate, time and cost-effective and has a positive effect on patient clinical management.[11],[12]

The range of applications of MALDI TOF MS has been rapidly increased to be used in the detection of resistance against different antimicrobial drugs.[13] The most common mechanism of resistance to lactams is the hydrolysis of the amide bond of the β-lactam ring. The hydrolysis degradation product shows a different molecular mass from that of a native molecule. MALDI TOF MS can detect carbapenemase activity in Gram-negative bacilli through the detection of the imipenem (IMP) and meropenem molecules and their natural degradation products. In September 2011, the first two studies on direct carbapenemase detection by MALDI TOF MS were reported.[14],[15] Here, we describe a method that can be used to detect carbapenemase activity among A. baumannii isolates in 3–4 h which when combined to rapid identification can save more than 24–30 h and therefore a better patient care.


 ~ Materials and Methods Top


Bacterial isolates and detection of carbapenamases

Fifty A. baumannii strains were used in this study; they originated from different clinical specimens of intensive care patients attending the Main University Hospital, Alexandria, Egypt, during a period of 6 months. Thirty-seven strains were isolated from bronchoalveolar lavage specimens; 6 from sputum, 6 from urine and one from blood culture. Strains were identified to the species level by the MALDI TOF MS using the Bruker Daltonics UltrafleXtreme Biotyper software (Bruker Daltonics, Bremen, Germany), as previously described.[16] Susceptibility of all isolates to IMP was determined, by the modified Bauer–Kirby disc diffusion technique, and interpreted in accordance with the guidelines established by the Clinical and Laboratory Standards Institute 2012 (CLSI).[17] Minimal inhibitory concentration (MIC) for IMP was performed by microbroth dilution technique as recommended by the CLSI, in which containers holding identical volume of broth with antimicrobial solution in incrementally increasing concentrations are inoculated with a known number of bacteria.[17] Class B MBLs were tested for using the IMP-ethylenediaminetetraacetic acid Combined Disc Synergy (ECDS) test.[18] As class A carbapenemases are inhibited by clavulanate, screening for production of Class A (Klebsiella pneumoniae carbapenemase [KPC]) carbapenemase was done using synergy test between amoxicillin-clavulanate (AMC) and carbapenems.[19]

MBL genes; blaIMP, blaVIM and blaSIM-1, and the OXA-carbapenemase genes; blaOXA-23, blaOXA-24 and blaOXA-58 were searched for by polymerase chain reaction.[20],[21]

Matrix-assisted laser desorption ionisation time of flight mass spectrometry analysis of imipenem

Commercial IMP (500 mg, cilastatin 500 mg) was obtained (Merck, Sharp and Dohme, USA) and diluted in 0.45% saline to a concentration of 2, 1, 0.5 and 0.25 mg/ml. Several matrices were tested (1) 10 mg/ml a-cyano-4 hydroxycinnamic acid (HCCA) in TA30 (TFA 0.1% 70: Acetonitrile 30); (2) 2,5-dihydroxybenzoic acid (DHB) diluted in acetonitrile and water (1/1) and (3) 3.3 mg/ml of HCCA diluted in a mixture of acetone, ethanol and TFA (1/2)[22] (matrices from Bruker Daltonics, all chemicals from Sigma Aldrich).

One microlitre of IMP solution was deposited on an Anchorchip384 target (Bruker Daltonics), allowed to dry at room temperature and then it was overlaid with 1 µl of each kind of matrix. Mass spectra were acquired with an UltrafleXtreme TOF/TOF MS and the FlexControl 3.0 software (Bruker Daltonics GmbH, Bremen, Germany) operating in positive reflection ion mode between m/z 0 and 1000 Da. The parameters were set as follows: Ion source 1: 25 kV; ion source 2: 21.5 kV; lens: 10 kV; reflector 1: 25.5 kV; reflector 2: 14.19 kV; pulsed ion extraction: 10 ns and detection gain: 9.16. A total of 3000 shots were acquired in six different positions for one spectrum.

Internal calibration of mass spectrometer

Peaks of Ser-His (243.10 Da), Lys-Lys-Lys (403.30 Da), Bradykinin (1-5) (573.31 Da) and Bradykinin (1-7) (757.39 Da) (MBT STAR ACS, Bruker Daltonics) were used for calibration.

Imipenem hydrolysis

A. baumannii strains were cultured overnight on blood agar plates at 37°C. A 1 µl loopful was added to 50 µl of 0.45% NaCl, with IMP concentrations ranging from 0.25 to 2 mg/ml. Bacteria and antibiotic mixtures were incubated at 37°C for 3 h, after which the tubes were centrifuged at 12,000 g for 5 min. One microlitre from the supernatant of each tube was applied on the target plate and overlaid with 1 µl matrix. Two spots for each strain were done in all experiments.[14]

Determination of carbapenamase activity by analysis of the produced spectra

For each spot, 3000 shots were acquired. Carbapenamase activity was positive if the IMP peak of 300 m/z disappeared completely after the incubation time or its intensity was reduced, with the appearance of a 254 m/z peak representing the IMP metabolite after hydrolysis with carbapenamases.


 ~ Results Top


Bacterial identification and antibiotic susceptibility

All isolates were identified as A. baumannii using the MALDI TOF MS technique, and all obtained scores more than 2.2 ensured excellent genus and species identification.

Microbroth dilution for IMP showed that out of the 50 isolates, 10 were susceptible with MIC between 1 and 4 µg/ml and the remaining 40 isolates were resistant with MIC ≥16 µg/ml, out of which, 10 isolates had MIC at 128 µg/ml or more indicating higher level of resistance to IMP. The susceptibility results of IMP by disc diffusion technique and microbroth dilution method (MIC) were similar, where ten isolates were susceptible by both techniques, forty isolates were resistant by both techniques.

Thirty-nine isolates were positive for the production of carbapenemases by modified Hodge test. The same 39 isolates were also positive for MBL production using the IMP-ECDS test and were negative for the synergy test between clavulanate (AMC) and carbapenems, excluding the production of class A KPC carbapenemases.

Detection of carbapenamase-encoding genes

Among the forty isolates resistant to IMP, one or more carbapenemase genes were detected [Table 1]. The most common gene was OXA23 as it was detected in 92% of these isolates, followed by VIM in 69%, SIM in 30.5% and finally IMP in 22% of these isolates. This shows that thirty isolates (84%) carried both class B and class D genes, while three isolates carried class B alone and three isolates carried class D alone.
Table 1: Distribution of the studied carbapenemase genes among Acinetobacter baumannii isolates according to the class of β-lactamase

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For the susceptible isolates, the most common gene was VIM (in 30%) followed by SIM and OXA23 (in 10%) [Table 1].

Standardisation of imipenem solution

The three matrices described in methods were tested with IMP and 0.45% saline. The HCCA in TA30 yielded the most comprehensive results, where the peaks for IMP and its metabolite appeared clearly without significant background peaks. This matrix was used for further tests. The stability of IMP was tested until 4 h from preparation, where IMP peaks were consistently at 300 m/z and no appearance of hydrolysis peaks.

Carbapenamase detection by imipenem hydrolysis assay

A. baumannii isolates producing carbapenamases were able to hydrolyse IMP [Figure 1] into its 254 m/z metabolite and concomitant disappearance or reduction in the intensity of the 300 m/z peak representing IMP [Figure 2]. Carbapenamase activity was considered when the ratio of the area under the curve between IMP and its metabolite was <0.5, and if more than 0.5, the strain was considered to be a nonproducer of carbapenamases [Supplementary Table S1 (Available online)].
Figure 1: Mass spectra of imipenem hydrolysis assay. The upper three panels (a-c) represent imipenem after a 3 h period of incubation with Acinetobacter baumannii imipenem-susceptible strains at 37°C, where intact imipenem appears at 300 m/z. The lower panels (d-f) represent imipenem after incubation with Acinetobacter baumannii-resistant strains for the same duration and temperature, where imipenem hydrolysis product appears at 254 m/z.

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Figure 2: Mass spectrum of imipenem, appears at 300 m/z as determined by UltrafleXtreme.

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These results were detected after a period of 3 h incubation using four different concentrations of IMP (0.25, 0.5, 1 and 2 mg/ml). The variation in the IMP concentration did not affect the test result, where the difference between the IMP peak and its metabolite was still significant with the four different concentrations.

All the fifty isolates were tested for carbapenamase production twice using the IMP hydrolysis assay, and the result was considered positive when there was a reduction or disappearance of the IMP 300.0 m/z peak and the appearance of the hydrolysed IMP at 254.0 m/z. The tested strains included 40 IMP-resistant A. baumannii strains, most of which harbour the blaOXA23 and/or VIM, SIM1 or IMP. For the resistant strains, carbapenamase activity was detected in 39 of the 40 IMP-resistant strains after 3 h incubation by disappearance of the 300.0 m/z peak and appearance of the 254.0 m/z peak with a ratio between the area under the peak of <0.5. The single IMP-resistant strain that did not show carbapenamase activity was also negative for the tested genes for carbapenamases and showed negative Hodge test and IMP-ECDS test, confirming that it is a noncarbapenamase producer and resistance to IMP which is probably due to other mechanisms. For the IMP-susceptible isolates, all strains were negative for carbapenamase production after 3 h by the presence of the 300.0 m/z peak and the ratio of the area of IMP and metabolite >0.5.

The sensitivity and specificity for carbapenamase production in the fifty isolates tested after 3 h incubation using 0.25 mg/ml IMP were 100% and 100%, respectively.


 ~ Discussion Top


The MALDI TOF MS in the past few years has created a revolutionary advance in microbiology towards fast, accurate, reliable and cost-effective techniques for identification of clinical isolates and recently for the determination of resistance to antimicrobial agents. This method was also applied for the diagnosis of not only bacteria but also for yeasts and mycobacteria.

In this study, MALDI TOF MS was used to detect the production of carbapenamases produced by A. baumannii strains isolated in Egypt. The carbapenamase production by these strains was evaluated by phenotypic assays including routine disc diffusion, MIC, special tests and genotypic assays for the detection of carbapenamase-encoding genes.

Recently, MALDI TOF MS was applied for the detection of carbapenamase activity in A. baumannii[22],[23],[24] and in Enterobacteriaceae, Pseudomonas aeruginosa and Bacteroides fragilis.[15],[25],[26],[27] Various carbapenems were used, such as IMP, meropenem and ertapenem. Here, we worked with IMP where it was stable in 0.45% saline all through the hours of incubation, and it did not require the use of Tris-HCL or SDS as reported for ertapenem and meropenem.

The mass MSs used up to date for the detection of carbapenamase activity include either the Microflex MALDI TOF [15], 23, [25],[26],[27] or Ultraflex I and Autoflex.[24] Here, we used the UltrafleXtreme which is more sensitive and provides greater resolution than the former instruments. Moreover, the use of the reflector mode for masses ranging from 0 to 1000 m/z provides greater resolution and mass accuracy. AncorChip target plates were used, where they have a hydrophilic centre or anchor surrounded by hydrophobic surroundings. This concentrates the sample and increases the detection sensitivity by a factor of 10–100 (Bruker Daltonics). Furthermore, the matrix plays an important role in the determination of the quality of spectra obtained by MALDI TOF, hence we tried three different matrix preparations described by Kempf et al.;[22] however, in our hands, the use of HCCA in TA30 was most superior to HCCA in a mixture of ethanol, acetone and TFA. Hrabak et al. reported that the matrix DHB in 50% ethanol was most suitable for the visualisation of meropenem in conjunction with a microflex MALDI TOF.[15]

The detection of carbapenamase activity after incubation of the bacteria with IMP was determined not only by the disappearance of the IMP peak at 300 m/z, but also by the appearance of a 254 m/z peak corresponding to IMP degradation product. It was noted that the 254 m/z peak may appear even in the absence of carbapenamases as a natural degradation process that happens in the presence of sodium chloride.[28] Therefore, we resorted to calculating the ratio between the areas under the curves of both peaks as described by Kempf et al.[22] This method was highly reproducible and simple as the IMP peaks were consistent and not associated with sodium salt variants as described in other studies.[23],[25],[26] Thus, this method can determine the presence of carbapenamases within 3 h and indicate resistance to carbapenems in general. However, absence of carbapenamase activity does not necessarily mean susceptibility of this isolate to carbapenems, as other nonenzymatic factors may present that require further phenotypic testing.

The use of MALDI TOF MS for the detection of carbapenamases is an assay that showed high sensitivity and specificity with no false-positive or false-negative results. Interestingly, there was one strain that was reported as a noncarbapenamase producer by MALDI TOF but was phenotypically resistant to IMP by disc diffusion and MIC tests. Genotypically, it did not possess any carbapenamase gene, implying the presence of other resistance mechanisms as efflux pumps or mutation of surface porins. That is why it is important to report isolates tested by MALDI TOF MS as carbapenamase producers or nonproducers rather than IMP susceptible or resistant, as this is not accurate and can be misleading to clinicians.


 ~ Conclusion Top


This assay has a very short turnaround time in comparison to other phenotypic assays, which will be of great benefit in all clinical settings where a quick decision is needed to start antibiotic therapy, especially since carbapenems are expensive drugs and precious at the same time, and the decision to administer them should be done with utmost care.

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

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2004 - Indian Journal of Medical Microbiology
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