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 ~  Abstract
 ~ Introduction
 ~ Subjects and Methods
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~  References
 ~  Article Figures
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 1  |  Page : 52-57
 

In vitro and in vivo fitness of clinical isolates of carbapenem-resistant and -susceptible Acinetobacter baumannii


1 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh; Department of Biotechnology, Maharishi Markandeshwar Deemed to be University, Mullana (Ambala), Haryana, India
2 Department of Microbiology, Government Medical College and Hospital, Sector -12, Chandigarh, India
3 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission12-Oct-2019
Date of Decision31-Mar-2020
Date of Acceptance26-Jun-2020
Date of Web Publication25-Jul-2020

Correspondence Address:
Dr. Vikas Gautam
Department of Medical Microbiology,Post Graduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_468

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 ~ Abstract 


Context: Acinetobacter baumannii is one among the leading nosocomial pathogens in the healthcare settings worldwide. Limited data on relative fitness and virulence of carbapenem-resistant A. baumannii (CRAB) are known. New methods are required to curb the rapidly rising antimicrobial resistance of this bug. Aims: We aimed to study the comparative in vitro and in vivo fitness of clinical isolates of CRAB and carbapenem-susceptible A. baumannii (CSAB). Settings and Design: A total of nine A. baumannii isolates were included in this study. CSAB ATCC-19606 was taken as a reference control strain. Subjects and Methods: Matrix-assisted laser desorption ionisation–time of flight mass spectrometry and gyrB and blaOXA-51PCR were used for species identification. Antimicrobial susceptibility was performed using Kirby-Bauer disk-diffusion method. Minimum inhibitory concentration for carbapenems (imipenem, meropenem and doripenem) was determined using agar dilution method. End point analysis, competitive index (CI), growth kinetics and generation time were determined for CRAB and CSAB isolates. In vivo fitness of CRAB and CSAB was determined using Caenorhabditis elegans host model. Multilocus sequence typing was performed to see the genetic relatedness of the isolates under study. Results: End point analysis, in vitro CI and growth kinetics experiments showed better fitness of clinical isolates of CRAB over CSAB ones. In vivo'nematode fertility assay' using C. elegans also supported the in vitro results. Conclusions: To the best of our knowledge, this is the first study of its kind from India showing difference in fitness of clinical isolates of CRAB and CSAB.


Keywords: Acinetobacter baumannii, Caenorhabditis elegans, carbapenem resistance, in vitro fitness


How to cite this article:
Kumar S, Singhal L, Ray P, Gautam V. In vitro and in vivo fitness of clinical isolates of carbapenem-resistant and -susceptible Acinetobacter baumannii. Indian J Med Microbiol 2020;38:52-7

How to cite this URL:
Kumar S, Singhal L, Ray P, Gautam V. In vitro and in vivo fitness of clinical isolates of carbapenem-resistant and -susceptible Acinetobacter baumannii. Indian J Med Microbiol [serial online] 2020 [cited 2020 Aug 11];38:52-7. Available from: http://www.ijmm.org/text.asp?2020/38/1/52/290678





 ~ Introduction Top


Acinetobacter baumannii is an important nosocomial pathogen, which causes an array of deadly hospital-acquired infections. Carbapenem-resistant A. baumannii (CRAB) has been reported as a threat to healthcare settings in developing countries.[1],[2] While carbapenems are considered as antibiotic of the last resort for a long time, the resistance rates of A. baumannii clinical isolates to these antimicrobials are increasing worldwide.[3],[4],[5] A recent study from Brazil indicated that the rates of infections caused by CRAB are increased by four times, especially in intensive care unit patients from 2013 to 2017.[6] Moreover, antimicrobial resistance in such microorganisms usually resulted in the treatment failure.[7] Over-expression of efflux pumps has various contrasting effects on bacterial cell fitness and their virulence potential. Recently, one study demonstrated the role of Ade RND efflux pumps in fitness and pathogenesis of A. baumannii.[8]

Fitness of a bacterium is a major physiological determinant, which is defined as the ability to modify the metabolism to survive and grow at different environmental conditions. There are different models to evaluate the fitness of bacteria such as determining the maximal growth rate in a batch culture.[9],[10],[11],[12] Alteration in the integrity of cell envelope is proved to activate the envelope stress response drastically, which significantly decreased the colonisation of A. baumannii.[13] Decreased virulence and lowin vivo bacterial fitness have also been found to be associated with resistance to colistin in A. baumannii.[14] During the last few decades, efforts have been made to understand the evolution and epidemiology of CRAB. Herein, we investigated the physiological properties to investigate the differential metabolic functions of CRAB and carbapenem-susceptible A. baumannii (CSAB) as opposed to genomic analysis. Globally, a very few studies have been previously attempted to study the relative fitness of A. baumannii. Therefore, in the present study, we investigated the correlations of carbapenem-resistant and -susceptible phenotypes of A. baumannii with their fitness and virulence potential usingin vitro andin vivo fitness experiments.


 ~ Subjects and Methods Top


Bacterial isolates, growth condition and species identification

A total of nine A. baumannii (5 CRAB and 4 CSAB) isolates were collected during 2013–2014 from the Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Isolates were collected from blood, sterile body fluids and cerebrospinal fluids. Samples were cultured on MacConkey and blood agar plates followed by incubation overnight at 37°C. Matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF-MS) was used to identify the isolate up to species level using isolated pure colonies.[15] Species identification was verified by detection of OXA-51 gene PCR and gyrB multiplex PCR.[16],[17] Genomic DNA was isolated using QIAamp genomic DNA kits (Qiagen). PCRs containing 2.5 mM dNTP mixture (Sigma Aldrich), 1 U (0.2 μl) of Taq polymerase (Sigma Aldrich), 10 pmol of each primer (IDT) and 1 μl of bacterial DNA were performed in a final volume of 25 μl. Following parameters were used for thermal cycling: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C–60°C for 30 s and 72°C for 1 min and final elongation at 72°C for 5 min. PCR products were analysed on agarose gel electrophoresis, stained with ethidium bromide and visualised on a ultraviolet transilluminator.

Drug-susceptibility profile

Kirby-Bauer disk-diffusion assay was used to determine the antimicrobial susceptibility of A. baumannii isolates for the following antibiotics; amikacin, ampicillin/sulbactam, ceftazidime, cefepime, ciprofloxacin, chloramphenicol, doripenem, gentamicin, imipenem, meropenem, piperacillin/tazobactam and tetracycline. The minimum inhibitory concentrations (MICs) of three carbapenems (imipenem, meropenem and doripenem) were determined using the agar dilution method for CRAB isolates [Table 1]. The CLSI guidelines were used to interpret the susceptibility results, according to the manufacturer's instructions.[18]
Table 1: Antimicrobial susceptibility (DD and minimum inhibitory concentrations) profile and multilocus sequence typing sequence types of Acinetobacter baumannii isolates

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Multilocus sequence typing

All the nine isolates of A. baumannii were subjected to multilocus sequence typing (MLST). Seven housekeeping genes (gltA, gdhB, gyrB, recA, gpi, cpn60 and rpoD) were amplified using PCR followed by sequencing of as previously described by Oxford scheme.[19],[20] PCR amplification was carried using reaction mixtures as stated in the first paragraph of methods. PCR profiles used for amplification are available on PubMLST database (https://pubmlst.org/abaumannii/info/primers_Oxford.shtml). The PCR products were subjected to 1.2% agarose gel electrophoresis followed by purification using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing primers were used for performing DNA sequencing by BigDye Terminator ready reaction mix, version 3.1 (Applied Biosystems, Waltham, MA, USA) in accordance with the manufacturer's protocol. The reaction products were analysed on an ABI PRISM genetic analyser 3100 (Applied Biosystems, Waltham, MA, USA). The sequences from both strands of each of seven loci of the same isolate were aligned separately, trimmed to the desired length and edited using SeqMan II program from the Lasergene software package (DNASTAR, Inc., Madison, WI, USA). Sequence types (STs), allele profiles and clonal complexes were assigned by PubMLST database (www.pubmlst.org/abaumannii/).

Determination of competitive in vitro fitness

End point analysis

Two isolates belonging to each of CSAB and CRAB were selected randomly. Each isolate was tested against each of the others for competitive growth in mixed culture (four sets). CSAB and CRAB were mixed in an equal ratio of 50:50 as stated previously.[10] Bacteria were cultured on Columbia sheep blood agar at 35°C for 24 h, followed by suspension in normal saline to obtain 1 McFarland standard density, and 30 μl of each suspension was inoculated into 3 ml of BHI broth followed by incubation at 37°C ± 1°C with gentle agitation using a magnetic stirrer. The number of viable cells was monitored at t = 0 and at stationary phase (t ≈ 18 h) by dilution plating in triplicate on MHA with or without carbapenems (imipenem – 6 μg/ml). The colony count was carried out after 18 h incubation at 37°C.[10]

In vitro competition index

Thein vitro competition index was determined from growth cultures that consisted of (1) ATCC-19606, (2) test strain and (3) mixed inocula of equivalent CFUs from both strains as dictated previously.[14] Bacteria at a concentration of 5 × 105 CFUs/ml were grown in 20 ml of BHI broth. At 0, 12, 24, 36, 48, 60 and 72 h, 100 μl aliquots were taken, and susceptible and resistant CFUs were counted by plating serial log dilutions on MHA with or without carbapenems (imipenem – 6 μg/ml). The competitive index (CI) was calculated as the number of test strain CFUs recovered/number of ATCC-19606 CFUs recovered, divided by the number of test strain CFUs inoculated/number of ATCC-19606 CFUs inoculated as mentioned previously.[14]

Growth kinetics

Representative isolates of CSAB and CRAB groups were selected randomly to study the kinetics of competitive growth. The isolates were grown on blood agar at 35°C for 24 h and inoculated at a density of 105 CFU/l into a 250 ml flask containing 200 ml of BHI broth as per already standardised protocol.[10] Sampling and inoculation were done through spinal needles in the caps of the flasks under sterile conditions. The flasks were incubated at 37°C ± 0.1°C in a thermostat-controlled water bath, and the medium was aerated using a magnetic stirrer. The number of viable cells was determined at each sampling time (1 h) for up to 10 h by plating two 100 μl portions of appropriate dilutions of the sample on blood agar with or without carbapenems (imipenem – 6 μg/ml). The ratio of CSAB and CRAB during growth was evaluated. This experiment was performed in triplicate.[10]

Generation time

The generation times of two randomly chosen CSAB and CRAB isolates were determined in BHI broth and compared with the resistance profile of the respective antibiotic susceptibility.

Nematode fertility assay

Fertility assays were used to analyse nematode fertility in the presence of different A. baumannii strains (CSAB and CRAB). Caenorhabditis elegans were grown to stage L1 and arrested overnight at 16°C to physiologically synchronise the nematodes. The L1 worms were cultivated in A. baumannii strain or Escherichia coli lawns to the L4 stage. The fertility assay was performed as described by Vallejo et al.[21] by inoculating a single L4 nematode per NGM plate (already containing the same A. baumannii strain or E. coli OP50) before incubation for 24 h. Over a period of 5 days, adult nematodes were removed daily to a fresh plate seeded with the same bacterial strain. To determine fertility, nematode progeny was counted daily, 48 h after parent removal. The assay was performed in triplicate.


 ~ Results Top


Bacterial identification

After initial Gram staining and biochemical reactions in the clinical bacteriology laboratory, bacterial isolates were identified as A. baumannii by MALDI-TOF-MS followed by confirmation of latter by using PCR amplification of blaOXA-51-like and gyrB genes. There was perfect harmony among different methods, which correctly identify the isolates as A. baumannii.

Antimicrobial-susceptibility testing

All the five CRAB isolates were observed as extensively drug resistant due to resistant to most of the antimicrobials used for susceptibility testing. All the four CSAB isolates were susceptible to all antimicrobials, except chloramphenicol and ceftazidime. Aside this, two CSAB isolates were resistant to cefotaxime and three were resistant to cefepime. MICs for three carbapenems (meropenem, imipenem and doripenem) were observed 32 μg/ml for all the five CRAB isolates [Table 1]. MICs for all the four CSAB were reported below <2 μg/ml [Table 1].

Multilocus sequence typing analysis

A total of seven different STs were observed in this study by MLST analysis. Three STs were previously reported (ST 20, ST 395 and ST 451) and four new STs (ST 1053, ST 1090, ST 1393 and ST 1394) were assigned to our isolates of A. baumannii. All three CSAB isolates showed new STs, whereas one of the CRAB isolate was assigned a new ST.

End point analysis

End point analysis showed that when two clinical isolates belonging to CRAB and CSAB groups were grown together for overnight, CRAB outgrew the susceptible one [Figure 1]. This experiment was performed utilising another set of clinical isolates with the same experimental conditions and reproduced the same results. However, when we used a typed strain ATCC-19606 A. baumannii, which was susceptible to carbapenems in competitive growth with clinical isolates of CRAB, the former strain showed a higher growth [Figure 1].
Figure 1: End point analysis showed ratios of CFUs of different combinations of carbapenem-susceptible to carbapenem-resistant isolates of Acinetobacter baumannii at time = 0 h and at time = 18 h

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In vitro competition index

A clinical isolate of CRAB 1507, when grown in competition with the CSAB ATCC-19606 strain, depicted better fitness than the latter. As per Monk et al., 2008,[22] CI >1 is considered as significant fitness and CRAB clinical isolate 1507 in this study showed a CI of 5.07, which was quite significant [Figure 2].
Figure 2: In vitro competition index of carbapenem-susceptible reference strain (CSAB-ATCC-19606) and carbapenem-resistant clinical isolate (CRAB-1507) of Acinetobacter baumannii

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Growth kinetics

Growth kinetic experiment also supported the better fitness of resistant isolate over susceptible ones. We performed this experiment utilising CRAB clinical isolate 1507 and CSAB strain ATCC-19606. These strains were grown separately under same experimental conditions, and the results showed that CRAB 1507 showed higher growth than CSAB ATCC-19606 [Figure 3]. However, both of these isolates showed no difference in the generation time.
Figure 3: Growth kinetics of carbapenem-susceptible reference strain (CSAB-ATCC 19606) and carbapenem-resistant clinical isolate (CRAB-1507) of Acinetobacter baumannii

Click here to view


Nematode fertility assay

There are very few studies on C. elegans being used as host infection model to study the virulence potential of A. baumannii.[23],[24] In the current study, C. elegans 'killing assay' was not able to distinguish this virulence difference and a similar study had been published previously.[21] However, we have distinguished CRAB isolates from CSAB ones by using fertility assay of C. elegans. CRAB isolates as checked byin vitro fitness experiments showed the higher virulence in C. elegans by reducing the number of progeny of C. elegans in comparison to CSAB ones and control strain E. coli Op50 [Figure 4].
Figure 4: Fertility assay showed fecundity rate of Caenorhabditis elegans on the overnight grown lawn of carbapenem-resistant and -susceptible isolates of Acinetobacter baumannii. Escherichia coli Op50 was used as control strain

Click here to view



 ~ Discussion Top


In the present study, we performedin vitro studies utilising different sets of experiments for the first time in clinical isolates of CRAB and CSAB. Inin vitro fitness study, resistant isolates showed better fitness than susceptible ones. This could be due to the acquisition of any new mutation in the resistant isolates, which enabled them to show better growth than susceptible ones. In the end point analysis, we observed that the resistant ones were more fit than susceptible ones among the clinical isolates, but the scenario of fitness was completely reversed when CSAB-ATCC-19606 was used with CRAB clinical isolates [Figure 1]. This could be due to difference in the origin and environment of the isolates. ATCC-19606 is the laboratory reference isolate, whereas it might be devoid of few mutations which are prevalent in the clinical isolates of patients' samples. In the beginning, many resistance mutations are not well adaptive, but most of these mutations can improve the fitness insufficiency. Finally, this progression can lead to fixation of resistance mutations and high fitness cost. However, no difference in the generation time was noticed among CSAB and CRAB isolates; remarkable differences in the fitness of CRAB and CSAB were noticed in growth kinetics andin vitro competition index experiments, where CRAB found out to be fitter. C. elegans fertility model showed more virulent CRAB over CSAB ones. MLST analysis showed circulation of different clones of A. baumannii in the hospital setting under study. It is interested to note that ST 451 was found only in CRAB isolates. Most of the new STs were found associated with CSAB isolates.

Till date, there are very few studies on the fitness associated with A. baumannii, although some newer studies are coming up with fitness associated with different parameters.[14] One recent study by Yoon et al. showed that utilisingin vitro competition experiments, overproduction of the Ade RND efflux pumps contributed to the considerable decrease in fitness of A. baumannii.[8] Smani et al. have studied for the first time the role of Omp33 in the fitness and virulence of A. baumannii.[25] Recently, changes in the cell envelope integrity are proved to activate the envelope stress response drastically, which significantly decreased the colonisation of A. baumannii.[13] Colistin resistance is also been studied and found to be associated with decreased virulence and lowin vivo bacterial fitness.[14] In the current study, CRAB isolates were found to be more fit and virulent than CSAB.

Several studies have suggested the involvement of genetic modifications in conferring antimicrobial resistance and in decreasing the growth rate by interfering natural physiological properties of bacteria.[26],[27],[28]In vitro fitness experiments offer a model to study and compare the fitness of bacteria. Bacterial fitness is prone to be modified in clinical practice without being noticed by clinicians and microbiologist. Such fitness advantages could possibly the reason of emergence of new clones with peculiar characteristics and resistance pattern.


 ~ Conclusions Top


To the best of our knowledge, this is the first study of its type in India where it was revealed that the resistance to carbapenems in A. baumannii was associated with higherin vitro bacterial fitness and virulence, dissimilar to other Gram-negative pathogens.In vivo experiments also showed the higher virulence potential of CRAB isolates using C. elegans infection model. Further, whole-genome sequencing-based genomic analysis of the regulatory network of efflux pumps could better reveal the fundamental mechanisms of antibiotic resistance in A. baumannii.

Financial support and sponsorship

The work was supported by PGIMER intramural grant.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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