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Mechanism of pathogenesis in multidrug resistant Acinetobacter baumannii isolated from intensive care unit
Gene Reports 18 (2020) 100557
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Mechanism of pathogenesis in multidrug resistant Acinetobacter baumannii isolated from intensive care unit
T
⁎
Saba Saadoon Khazaala, Israa M.S. Al-Kadmya,b, , Sarah Naji Aziza a b
Department of Biology, College of Science, Mustansiriyah University, Baghdad POX10422, Iraq Faculty of science & Engineering, School of Engineering, University of Plymouth, PL4 8AA, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: Acinetobacter baumannii Virulence Pathogenesis Proteomic analysis
Background: This work examined potent virulent A. baumannii isolates through proteomic and transcriptional analyses to demonstrate multi-factorial virulence and pathogenicity of this organism to growth within its host. Methods: We analyzed a clinical isolates of A. baumannii named ISR0031 through proteomic SDS-PAGE and microarray to identify the differentially expressed protein and genes in comparison to an ATCC 17978. Results: A total of 23 proteins were identified in SDS-PAGE includes from protein Synthesis pathway, membrane In microarray analyses 135 genes were found to be differentially regulated in ISR0031 among which majority of genes were involved in secondary metabolite biosynthesis, transport, catabolism, and transcriptional regulation. Conclusions: Up-regulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics and that is the major cause in A. baumannii explained how it poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane.
1. Background Infections remain a significant challenge in intensive care unit despite promising advances in medical care. A. baumannii is among the most frequent causes of infection which has substantially gained increasing recognition as a cause of nosocomial infections. It has well established itself in hospital niche being responsible for approximately 20% ICU infections worldwide (Vincent et al., 2009). A. baumannii infection is pronounced predominantly in the developing world due to high resistant rate and it is become of one of three main causes of nosocomial pneumonia and blood stream infections (Allegranzi et al., 2011). A. baumannii is an opportunistic pathogen that rarely causes disease in healthy individuals. An extensive regulatory capacity is encoded within the genome of A. baumannii, which likely contributes to this organism's ability to adapt to a broad range of environmental conditions. Despite having known for its lethal role in immunocompromised patients this organism has not known for demonstrating its virulent pathology, and elaborates very few known virulence factors. Few known among virulence factors are outer Membrane Protein A (OmpA), Penicillin Binding Protein and Phospholipase D (Pld). There are few others (Choi et al., 2008; Jacobs et al., 2010; Russo et al., 2008). Putative virulence factors such as iron acquisition systems and genes involved in biofilm formation, but not all of them have demonstrated ⁎
their roles in vivo (Rodriguez-Bano et al., 2008; Queenan et al., 2012; Brandtzaeg et al., 1995; Dorsey et al., 2004; Gaddy et al., 2009; Lee et al., 2008). This work represents progress toward understanding A. baumannii pathogenesis. In addition to identifying virulence factors, investigating the interaction of bacterial factors with the host is fundamental to our understanding of the pathogenic mechanisms employed by A. baumannii. Defining the host response to A. baumannii is critical to understanding A. baumannii pathogenesis. Although much attention has focused on how the innate immune response contributes to clearance of A. baumannii, evidence exists in the literature that the inflammatory response may also contribute to pathogenesis. This study aimed to examine potent virulent A. baumannii isolates through proteomic and transcriptional analyses which would help us to demonstrate that among clinical isolates what all multi-factorial regulation control the virulence and pathogenicity of this organism and provide the suitability of this organism to growth within the hospital environment and within its host. 2. Methods 2.1. Ethical statements for human/animal experiments The study was approved by institutional ethics committee and informed constant were obtained by each individual participants. Each
Corresponding author at: Department of Biology, College of Science, Mustansiriyah University, Baghdad POX10422, Iraq. E-mail address: [email protected] (I.M.S. Al-Kadmy).
https://doi.org/10.1016/j.genrep.2019.100557 Received 7 October 2019; Received in revised form 1 November 2019; Accepted 7 November 2019 Available online 14 November 2019 2452-0144/ © 2019 Published by Elsevier Inc.
Gene Reports 18 (2020) 100557
S.S. Khazaal, et al.
participant was known about the study follow up before enrolling for the study. I confirm that all experiments were performed in accordance with relevant guidelines and regulations. The reported study was approved from the institutional ethical review board. In this report we have analyzed A. baumannii, a clinical isolates. The isolate was acquired from patients admitted to intensive care units (ICU) of tertiary care referral hospital in the Baghdad state of IRAQ who was infected frequently with A. baumannii between March and October 2017 were included in this study. Individual was tested positive for three earlier instant in same period when evaluated in outdoor clinics and the isolates was obtained during his admission to ICU positive for infection less than 48 h after admission were excluded. The isolates was labelled as ISR0031, was identified as A. baumannii strain which was carbapenemase-producing strain with an imipenem MIC of > 16 μg/ml and meropenem MIC > 32 μg/ml. A. baumannii ATCC 17978 was used in each experiment as control to be compared with test results.
(20 mg/ml) (QIAGEN, Valencia, CA). Total RNA was extracted Mini columns (using Qiagen RNeasy®) from lysate following the manufacturer's instruction as recommendation QIAGEN for prokaryotic RNA purification. The GeneChips® was custom designed in this study using the genomic sequence of A. baumannii strain ATCC 17978 and all additional gene entries available at the time of design (270). On gene chips, 3891 predicted open reading frames and 3928 intergenic regions (> 50 base pairs) were represented. Following the manufacturer's recommendations and instruction 10 μg RNA sample was subjected to reverse transcription, followed by its conjugation with 3′ biotinylated, and hybridization to a GeneChip®, prepared particularly for A. baumannii antisense prokaryotic arrays (Affymetrix, CA, USA). cDNA (2 μg) was hybridized to 2 GeneChip according to the manufacturer's instruction and data were analyzed as described in Hood et al. (2010). Genes that were considered differentially expressed in either of test or ≥2-fold increase or decrease, were determined by Student's t-test and considered significant changed expression if p was determined ≤0.05.
2.2. SDS-PAGE analysis of supernatant proteins
3. Results
Isolate ISR0031 was grown overnight in Luria Broth (LB) and incubated at 37 0C with shaking at 210 rpm. Bacterial cells were harvested using centrifugation, and it was filtered through syringe filters (0.22 μm, Millipore Corporation, Merck) to obtain a cell free extract. Supernatant were diluted four fold with adding of cold trichloroacetic acid (TCA) at 40 °C to obtain a final concentration of 20% (v/v) to acquire proteins which were precipitated at 4 °C overnight. Precipitated proteins were collected by centrifugation and washed in cold ethanol (95%). Pelles was resuspended in Laemmli sample buffer (Suzuki et al., 1998) and run in SDS-PAGE using 20% polyacrylamide gels and was visualized using coomassie Brilliant Blue stain (Pierce, Rockford, IL).
3.1. A. baumannii ISR0031 secretes virulence determinants more compare to ATCC The purpose to investigate the proteomic profile of clinical isolate was to compare its virulence determinants to that A. baumannii ATCC that may be secreted more or less within the host environment upon infection. The supernatant protein of liquid culture of clinical isolates and ATCC strain was compared (Fig. 1). To normalized the protein content of sample and rule out difference of cell lysis of membrane, the whole protein content were measured in sample and propidium iodidestaining of cells were taken by flow cytometric analyses which confirmed no significant difference in membrane-compromised to membrane intact cells (data not shown). Proteins were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and searching sequences through database resulted in identification of approximately 60 proteins in A. baumannii supernatants (Table 1). A total of 23 proteins from different domain were identified from the band of SDS-PAGE. These proteins were mainly from protein Synthesis pathway, membrane Secreting protein, and efflux protein. A good number of proteins identified in SDS-PAGE were differentially secreted in ISR0031 (Table 1). Outer membrane proteins dominated the samples in both and overrepresented significantly in ISR0031, while few of efflux protein such as 33–36 kDa
2.3. Identification of protein bands The gel band were analyzed and those with visible clearly dense in either of control or test sample were excised for further process. In brief, the gel bands regions were excised after it was washed with ammonium bicarbonate (100 Mm) and treated with DTT (5 mM) to reduce the. Iodoacetamide (10 mM) was added following that it was washed 50 mM ammonium bicarbonate and dehydration with 100% acetonitrile. The gel pieces were completely dried, and digested with trypsin solution (Promega, Madison, WI) overnight at 37 °C. Peptides were removed using 60% acetonitrile/0.1% trifluoroacetic acid, and dried in vacuo. Samples were subjected to LC-MS analysis. 2.4. LC-MS-MS Analysis and Protein Identification Peptides were identified in a LTQ ion trap instrument (Thermofisher, LTQ) fortified with Surveyor HPLC pump (Thermofisher), Nanospray source, MicroAS autosampler and Xcalibur 2.0 SR2 control. Before running under LTQ-Ion trap sample were processed according to instruction and separated on liquid silicate similar to that previously described. The samples were run in to the injection to attain 700 nL flow-rates using mobile phase which contain 0.1% formic acid in acetonitrile at 400–2000 m/z with over nine scans (MS/MS). Tandem spectra were acquired and identity of proteins was searched through UniRef100 database using the SEQUEST algorithm. 2.5. Microarray analysis based on gene chips Overnight cultures of Isolate ISR0031 and A. baumannii ATCC adjusted to 0.5 OD in fresh LB medium then diluted up to two fold with ice-cold ethanol:acetone. Total RNA of sample was extracted following standard protocol earlier described (Janke et al., 2001). In brief, cells pellets were collected by centrifugation and disrupted by enzymatic lysis using lysozyme (10 mg/mL) in TE buffer containing proteinase K
Fig. 1. comparison between the supernatant protein of liquid culture of clinical isolates and ATCC strain. 2
Gene Reports 18 (2020) 100557
S.S. Khazaal, et al.
Table 1 The SDS-PAGE indicating the proteins which were differentially expressed in ISR0031, a clinically originated isolates. S. no
Description
ISR0031
ATCC
p-Value
Predicted MW (kDa)
Protein synthesis/chaperone proteins 1 Elongation factor 60 kDa chaperonin 2 Chaperone protein dnaK 3 Thiol:disulfide interchange protein
0.212 0.219 0.278
0.195 0.231 0.13866667
0.781 0.848 0.31433333
60.5 62.6 47
Membrane secreating protien 4 Outer membrane protein A 5 Putative outer membrane protein 6 Outer-membrane lipoproteins carrier protein 7 Peptidoglycan-associated lipoprotein 8 Lipoprotein
6.1705 2.997 3.0039 2.9716 0.5206
1.00375 0.7225 0.7467 0.3971 0.209
0.02025 0.04175 0.0798 0.0608 0.3629
38.4 37.05 25.091 20.82 29.88
Electron transport chain 9 ATP synthase subunit beta 10 ATP synthase subunit alpha
4.64196 0.11009
0.86355 0.1919
0.02525 0.02323
33.09 51.2
Bacterial programmed cell death 11 Bacteriocin (entericidin)
0.416
0.127
0.2
50.2
Cell wall biosynthesis 12 Murein lytic transglycosylase, soluble
0.685
0.48166667
0.32533333
53.76666667
Efflux protein 13 14 15 16 17 18
0.6095 0.3065 0.592 0.7015 1.23825 0.70725
2.859 0.1775 0.124 0.167 0.52575 0.2055
0.067 0.2095 0.0215 0.0385 0.1515 0.0725
35.35 66.65 42.15 37.7 29.025 20.625
Glucose metabolism 19 Enolase
2.01475
1.243
0.07875
38.875
Other 20 21 22 23
0.29304 1.20435 1.02342 0.16095
0.4551 0.71928 0.9546 0.25752
0.02775 0.78255 0.85914 0.33411
21.05 45.99 19.01 38.1
33–36 kDa outer membrane protein - associated beta-lactamase (AmpC) RND family drug transporter (AdeK) RND family drug transporter (AdeK) Putative signal peptide, metallo-beta-lactamase Putative signal peptide OsmY
Alkyl hydroperoxide reductase, C22 subunit Protein TolB precursor CsuA/B RecA
medical fraternity. A. baumannii sps have shown potential to represents the mounting burden of infections and also has proven challenging in the hospital due to its ability to persist (Queenan et al., 2012; Chan et al., 2007). Many a studies has established its virulence determinate using in-vitro approaches but those are yet to be verified in human model. This studies uses a proteomic approaches to identify the virulence factors and gene present in a clinical isolates ISR0031 of A. baumannii and compared their expression with A. baumannii strain ATCC 17978. The most important finding of these studies was 33–36 kDa outer membrane protein (omp), which is a porins having a major role in antibiotic transport through the outer membrane (CarO) was actively expressed in ATCC but lost the similar level of expression in ISR0031 which might be associated with resistance to carbapenems (Siroy et al., 2005). Study also observed the up-regulation of several genes associated with protein synthesis, membrane secreting protein and efflux protein. Few outer membrane proteins and gene associated with antibiotic resistance suggests that during the infection A. baumannii may have an impact on antibiotic resistance. Microarray analyses highlighted the differential expression of 36 gene representing include the membrane fusion AdeA efflux pump (AdeA) (Hood et al., n.d.; Magnet et al., 2001), which mediates resistance to a number of antibiotics in A. baumannii. Expression of this gene was shot up to 25 times folds than ATCC stain. Proteomic analyses highlighted an increased abundance of ABC transporter which is essential in cell viability, virulence, and pathogenicity. ABC trabsporters are supportive for iron ABC uptake systems, which are important effectors of virulence. The rise of extensively virulent A. baumannii is not witnessed so far but the growth in drug resistant capable organism in this species causes lethal infections upsurging a public health crisis particular challenge due to its adaptability within the hospital and host environments and the ability to readily
associated with membrane, signal peptide were major significant one. ATP synthase subunit beta was another major under electron transporter chain which was over expressed in clinical isolates. Proteomic analyses of supernatant proteins writes the details of release of proteins into culture media and determine if there is significant transcriptionally mediated changes in these two isolates. These was determined by microarray analyses using RNA isolated from stationary culture of bacteria and analyzed under hybridization of Affymetrix GeneChip® arrays. Total of 135 genes were found to be differentially regulated in ISR0031 (Table 2). Majority of genes were found to be involved in secondary metabolite biosynthesis, catabolism, transport and transcriptional regulation. There were seven major protein which were up regulated ten folds in ISR0031, as listed here-AdeA membrane fusion protein, putative ferric acinetobacter in transport system, Multidrug efflux transport protein, heavy metal RND efflux outer membrane protein (CzcC) putative ABC transporter, putative ABC-type antimicrobial peptide transport system, ABC-type nitrate/ sulfonate/bicarbonate transport systems. Interestingly, we observed down-regulation of AdeK proteins same time. (Table 2) and constitutive expression of OmpA, and AmpC, all of these were increased in culture supernatants of ISR0031. We also noticed that few proteins that increased in abundance in culture supernatants did not show much significant changes at transcript levels, indicating a possibility these proteins might be representing a post-translational mechanism for down regulation of target.
4. Discussion Acinetobacter baumannii is nosocomial pathogens and mainly upsurge pathogenesis in intensive care unit patients. A. baumannii is symbolic of the impending public health emergency threatening the 3
Gene Reports 18 (2020) 100557
S.S. Khazaal, et al.
5. Conclusions
Table 2 Proteomic approaches (microarray) analysis showed the genes differentially expressed in. S. no
Description
Fold-induction
p-Value
1 2
AdeA membrane fusion protein Putative ferric acinetobactin transport system Multidrug efflux transport protein Heavy metal RND efflux outer membrane protein (CzcC) Putative ABC transporter Putative ABC-type antimicrobial peptide transport system ABC-type nitrate/sulfonate/bicarbonate transport systems Outer membrane protein A ATP synthase subunit beta Transporter, LysE family Putative RND family cation/multidrug efflux pump Putative MFS transporter Outer-membrane lipoproteins carrier protein Peptidoglycan-associated lipoprotein Putative RND family drug transporter Major facilitator superfamily Heavy metal efflux pump (CzcA) Potassium-transporting ATPase A chain 33–36 kDa outer membrane protein associated Putative transporter DMT family permease Enolase Putative ATP-binding component of ABC transporter Putative signal peptide, metallo-betalactamase Protein TolB precursor CsuA/B Putative signal peptide OsmY RND family drug transporter (AdeK) Bacteriocin (entericidin) Beta-lactamase (AmpC) Alkyl hydroperoxide reductase, C22 subunit Thiol:disulfide interchange protein Chaperone protein dnaK Elongation factor 60 kDa chaperonin RecA ATP synthase subunit alpha
25.9 23.2
0.065975 0.137865
17.6 16
0.0211393 0.0018225
14.3 11.8
0.019565 0.77168
10.1
0.0020907
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
6.1705 4.64196 3.9 3.2
0.0716625 0.0229775 0.019565 0.0037575
3.2 3.0039
0.072618 0.7121205
2.9716 2.9 2.7 2.6 2.4 2.2585
0.7818174 0.07029 0.055328 0.02829 0.190645 0.067
2.2 2.1 2.01475 2
0.0379925 0.007182 0.07875 0.190645
1.23825
0.1515
1.20435 1.02342 0.70725 0.7015 0.416 0.3065 0.29304 0.278 0.219 0.212 0.16095 0.11009
0.78255 0.85914 0.0725 0.0385 0.2 0.2095 0.02775 0.065975 0.137865 0.035035 0.33411 0.02323
Up-regulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics and that is the major cause in A. baumannii explained how it poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane. Author contribution All authors contributed equally to the Literature review, Study plan, Lab work, fund generation, Manuscript writing, analyses data and proof. Declaration of competing interest The authors declare no conflict of interest. The authors themselves are responsible for the content and writing of the paper. Acknowledgement The authors would like to thank Mustansiriyah University (https:// uomustansiriyah.edu.iq/)Baghdad, Iraq for its support to complete this work. Ethical statements for human/animal experiments The study was approved by institutional ethics committee “University of Mustansiriyah” and informed consent was obtained in written by each individual participants. Each participant was known about the study follow up before enrolling for the study. I confirm that all experiments were performed in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and material All data available in manuscript. References
acquire resistance (Magnet et al., 2001; Marchand et al., 2004; Peleg et al., 2007). Similar to mammals, pathogens have evolved to acquire the defenses and proliferation results in strains that modify the host response to bring down pathogen clearance. The precise mechanism for attenuating such effect in this organism is not known. The results from these studies are very evident that a number of transporters like ABC, RND family efflux pump, Ade A and Ade K expression were differentially expressed in clinical isolates. These transporters have been investigate for their role in bacteria and its established fact that they have big role to play for bacterial resistant abilities (Pournaras et al., 2006; Al-Kadmy et al., 2018; Kareem et al., 2017; AL_Jubori et al., 2016). Multidrug efflux transporters were another important gene boosted in the clinical isolated almost 17 folds. This is a powerful mechanism extrude a number of structurally unrelated antibiotics classes from the bacterial cell, also includes toxic heavy metal ions, assisting their survival in harmful environments. Furthermore, upregulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics (Peleg et al., 2007; Pournaras et al., 2006; Kareem et al., 2017; AL_Jubori et al., 2016). A. baumannii poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane.
AL_Jubori, S.S., AL_Kadmy, I.M.S., Al_Ani, Z.J., 2016. Emergence of multidrug resistance (MDR) Acinetobacter baumannii isolated from Iraqi hospitals. Adv. Environ. Biol. 10, 265–276. Al-Kadmy, I.M.S., Ali, A.N.M., Salman, I.M.A., Khazaal, S.S., 2018. Molecular characterization of Acinetobacter baumannii isolated from Iraqi hospital environment. New Microbes New Infect. 21, 51–57. Allegranzi, B., Bagheri Nejad, S., Combescure, C., Graafmans, W., Attar, H., Donaldson, L., Pittet, D., 2011. Burden of endemic health-care-associated infection in developing countries: systematic review and meta-analysis. Lancet 377, 228–241. Brandtzaeg, P., Gabrielsen, T.O., Dale, I., Müller, F., Steinbakk, M., Fagerhol, M.K., 1995. The leucocyte protein L1 (calprotectin): a putative nonspecific defence factor at epithelial surfaces. Adv. Exp. Med. Biol. 371A, 201–206. Chan, P.-C., Huang, L.-M., Lin, H.-C., Chang, L.-Y., Chen, M.-L., Lu, C.-Y., Lee, P.-I., Chen, J.-M., Lee, C.-Y., Pan, H.-J., Wang, J.-T., Chang, S.-C., Chen, Y.-C., 2007. Control of an outbreak of pandrug-resistant Acinetobacter baumannii colonization and infection in a neonatal intensive care unit. Infect. Control Hosp. Epidemiol. 28, 423–429. Choi, C.H., Lee, J.S., Lee, Y.C., Park, T.I., Lee, J.C., 2008. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol. 8, 216. Dorsey, C.W., Tomaras, A.P., Connerly, P.L., Tolmasky, M.E., Crosa, J.H., Actis, L.A., 2004. The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related. Microbiology 150, 3657–3667. Gaddy, J.A., Tomaras, A.P., Actis, L.A., 2009. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect. Immun. 77, 3150–3160. Hood, M.I., Jacobs, A.C., Sayood, K., Dunman, P.M., Skaar, E.P., 2010. Acinetobacter baumannii increases tolerance to antibiotics in response to monovalent cations.
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resistant Acinetobacter baumannii isolates expressing OXA-58 carbapenemase in an intensive care unit. J. Antimicrob. Chemother. 57, 557–561. Queenan, A.M., Pillar, C.M., Deane, J., Sahm, D.F., Lynch, A.S., Flamm, R.K., Peterson, J., Davies, T.A., 2012. Multidrug resistance among Acinetobacter spp. in the USA and activity profile of key agents: results from CAPITAL Surveillance 2010. Diagn. Microbiol. Infect. Dis. 73, 267–270. Rodriguez-Bano, J., Marti, S., Soto, S., Fernandez-Cuenca, F., Cisneros, J.M., Pachon, J., Pascual, A., Martinez-Martinez, L., McQueary, C., Actis, L.A., Vila, J., 2008. Biofilm formation in Acinetobacter baumannii: associated features and clinical implications. Clin. Microbiol. Infect. 14, 276–278. Russo, T.A., Beanan, J.M., Olson, R., Macdonald, U., Luke, N.R., Gill, S.R., Campagnari, A.A., 2008. Rat pneumonia and soft-tissue infection models for the study of Acinetobacter baumannii biology. Infect. Immun. 76, 3577–3586. Siroy, A., Molle, V., Lemaitre-Guillier, C., Vallenet, D., Pestel-Caron, M., Cozzone, A.J., Jouenne, T., De, E., 2005. Channel formation by CarO, the carbapenem resistanceassociated outer membrane protein of Acinetobacter baumannii. Antimicrob. Agents Chemother. 49, 4876–4883. Suzuki, K., Suzuki, M., Taiyoji, M., Nikaidou, N., Watanabe, T., 1998. Chitin binding protein (CBP21) in the culture supernatant of Serratia marcescens 2170. Biosci. Biotechnol. Biochem. 62 (1), 128–135. Vincent, J.-L., Rello, J., Marshall, J., Silva, E., Anzueto, A., Martin, C.D., Moreno, R., Lipman, J., Gomersall, C., Sakr, Y., Reinhart, K., Investigators, E.I.G., 2009. International study of the prevalence and outcomes of infection in intensive care units. JAMA 302, 2323–2329.
Antimicrob. Agents Chemother. 54, 1029–1041. Jacobs, A.C., Hood, I., Boyd, K.L., Olson, P.D., Morrison, J., Carson, S., Sayood, K., Iwen, P.C., Skaar, E.P., Dunman, P.M., 2010. Inactivation of phospholipase D diminishes Acinetobacter baumannii pathogenesis. Infect. Immun. 78, 1952–1962. Janke, J., Engeli, S., Gorzelniak, K., Sharma, A.M., 2001. Extraction of total RNA from adipocytes. Horm. Metab. Res. 33 (4), 213–215 Apr. Kareem, S.M., Al-Kadmy, I.M.S., Al-Kaabi, M.H., Aziz, S.N., Ahmad, M., 2017. Acinetobacter baumannii virulence is enhanced by the combined presence of virulence factors genes phospholipase C (plcN) and elastase (lasB). Microb. Pathog. 110, 568–572. https://doi.org/10.1016/j.micpath.2017.08.001. Lee, H.W., Koh, Y.M., Kim, J., Lee, J.C., Lee, Y.C., Seol, S.Y., Cho, D.T., 2008. Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clin. Microbiol. Infect. 14, 49–54. Magnet, S., Courvalin, P., Lambert, T., 2001. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob. Agents Chemother. 45, 3375–3380. Marchand, I., Damier-Piolle, L., Courvalin, P., Lambert, T., 2004. Expression of the RNDtype efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS twocomponent system. Antimicrob. Agents Chemother. 48, 3298–3304. Peleg, A.Y., Adams, J., Paterson, D.L., 2007. Tigecycline efflux as a mechanism for non susceptibility in Acinetobacter baumannii. Antimicrob. Agents Chemother. 51, 2065–2069. Pournaras, S., Markogiannakis, A., Ikonomidis, A., Kondyli, L., Bethimouti, K., Maniatis, A.N., Legakis, N.J., Tsakris, A., 2006. Outbreak of multiple clones of imipenem-
5
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Mechanism of pathogenesis in multidrug resistant Acinetobacter baumannii isolated from intensive care unit
T
⁎
Saba Saadoon Khazaala, Israa M.S. Al-Kadmya,b, , Sarah Naji Aziza a b
Department of Biology, College of Science, Mustansiriyah University, Baghdad POX10422, Iraq Faculty of science & Engineering, School of Engineering, University of Plymouth, PL4 8AA, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: Acinetobacter baumannii Virulence Pathogenesis Proteomic analysis
Background: This work examined potent virulent A. baumannii isolates through proteomic and transcriptional analyses to demonstrate multi-factorial virulence and pathogenicity of this organism to growth within its host. Methods: We analyzed a clinical isolates of A. baumannii named ISR0031 through proteomic SDS-PAGE and microarray to identify the differentially expressed protein and genes in comparison to an ATCC 17978. Results: A total of 23 proteins were identified in SDS-PAGE includes from protein Synthesis pathway, membrane In microarray analyses 135 genes were found to be differentially regulated in ISR0031 among which majority of genes were involved in secondary metabolite biosynthesis, transport, catabolism, and transcriptional regulation. Conclusions: Up-regulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics and that is the major cause in A. baumannii explained how it poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane.
1. Background Infections remain a significant challenge in intensive care unit despite promising advances in medical care. A. baumannii is among the most frequent causes of infection which has substantially gained increasing recognition as a cause of nosocomial infections. It has well established itself in hospital niche being responsible for approximately 20% ICU infections worldwide (Vincent et al., 2009). A. baumannii infection is pronounced predominantly in the developing world due to high resistant rate and it is become of one of three main causes of nosocomial pneumonia and blood stream infections (Allegranzi et al., 2011). A. baumannii is an opportunistic pathogen that rarely causes disease in healthy individuals. An extensive regulatory capacity is encoded within the genome of A. baumannii, which likely contributes to this organism's ability to adapt to a broad range of environmental conditions. Despite having known for its lethal role in immunocompromised patients this organism has not known for demonstrating its virulent pathology, and elaborates very few known virulence factors. Few known among virulence factors are outer Membrane Protein A (OmpA), Penicillin Binding Protein and Phospholipase D (Pld). There are few others (Choi et al., 2008; Jacobs et al., 2010; Russo et al., 2008). Putative virulence factors such as iron acquisition systems and genes involved in biofilm formation, but not all of them have demonstrated ⁎
their roles in vivo (Rodriguez-Bano et al., 2008; Queenan et al., 2012; Brandtzaeg et al., 1995; Dorsey et al., 2004; Gaddy et al., 2009; Lee et al., 2008). This work represents progress toward understanding A. baumannii pathogenesis. In addition to identifying virulence factors, investigating the interaction of bacterial factors with the host is fundamental to our understanding of the pathogenic mechanisms employed by A. baumannii. Defining the host response to A. baumannii is critical to understanding A. baumannii pathogenesis. Although much attention has focused on how the innate immune response contributes to clearance of A. baumannii, evidence exists in the literature that the inflammatory response may also contribute to pathogenesis. This study aimed to examine potent virulent A. baumannii isolates through proteomic and transcriptional analyses which would help us to demonstrate that among clinical isolates what all multi-factorial regulation control the virulence and pathogenicity of this organism and provide the suitability of this organism to growth within the hospital environment and within its host. 2. Methods 2.1. Ethical statements for human/animal experiments The study was approved by institutional ethics committee and informed constant were obtained by each individual participants. Each
Corresponding author at: Department of Biology, College of Science, Mustansiriyah University, Baghdad POX10422, Iraq. E-mail address: [email protected] (I.M.S. Al-Kadmy).
https://doi.org/10.1016/j.genrep.2019.100557 Received 7 October 2019; Received in revised form 1 November 2019; Accepted 7 November 2019 Available online 14 November 2019 2452-0144/ © 2019 Published by Elsevier Inc.
Gene Reports 18 (2020) 100557
S.S. Khazaal, et al.
participant was known about the study follow up before enrolling for the study. I confirm that all experiments were performed in accordance with relevant guidelines and regulations. The reported study was approved from the institutional ethical review board. In this report we have analyzed A. baumannii, a clinical isolates. The isolate was acquired from patients admitted to intensive care units (ICU) of tertiary care referral hospital in the Baghdad state of IRAQ who was infected frequently with A. baumannii between March and October 2017 were included in this study. Individual was tested positive for three earlier instant in same period when evaluated in outdoor clinics and the isolates was obtained during his admission to ICU positive for infection less than 48 h after admission were excluded. The isolates was labelled as ISR0031, was identified as A. baumannii strain which was carbapenemase-producing strain with an imipenem MIC of > 16 μg/ml and meropenem MIC > 32 μg/ml. A. baumannii ATCC 17978 was used in each experiment as control to be compared with test results.
(20 mg/ml) (QIAGEN, Valencia, CA). Total RNA was extracted Mini columns (using Qiagen RNeasy®) from lysate following the manufacturer's instruction as recommendation QIAGEN for prokaryotic RNA purification. The GeneChips® was custom designed in this study using the genomic sequence of A. baumannii strain ATCC 17978 and all additional gene entries available at the time of design (270). On gene chips, 3891 predicted open reading frames and 3928 intergenic regions (> 50 base pairs) were represented. Following the manufacturer's recommendations and instruction 10 μg RNA sample was subjected to reverse transcription, followed by its conjugation with 3′ biotinylated, and hybridization to a GeneChip®, prepared particularly for A. baumannii antisense prokaryotic arrays (Affymetrix, CA, USA). cDNA (2 μg) was hybridized to 2 GeneChip according to the manufacturer's instruction and data were analyzed as described in Hood et al. (2010). Genes that were considered differentially expressed in either of test or ≥2-fold increase or decrease, were determined by Student's t-test and considered significant changed expression if p was determined ≤0.05.
2.2. SDS-PAGE analysis of supernatant proteins
3. Results
Isolate ISR0031 was grown overnight in Luria Broth (LB) and incubated at 37 0C with shaking at 210 rpm. Bacterial cells were harvested using centrifugation, and it was filtered through syringe filters (0.22 μm, Millipore Corporation, Merck) to obtain a cell free extract. Supernatant were diluted four fold with adding of cold trichloroacetic acid (TCA) at 40 °C to obtain a final concentration of 20% (v/v) to acquire proteins which were precipitated at 4 °C overnight. Precipitated proteins were collected by centrifugation and washed in cold ethanol (95%). Pelles was resuspended in Laemmli sample buffer (Suzuki et al., 1998) and run in SDS-PAGE using 20% polyacrylamide gels and was visualized using coomassie Brilliant Blue stain (Pierce, Rockford, IL).
3.1. A. baumannii ISR0031 secretes virulence determinants more compare to ATCC The purpose to investigate the proteomic profile of clinical isolate was to compare its virulence determinants to that A. baumannii ATCC that may be secreted more or less within the host environment upon infection. The supernatant protein of liquid culture of clinical isolates and ATCC strain was compared (Fig. 1). To normalized the protein content of sample and rule out difference of cell lysis of membrane, the whole protein content were measured in sample and propidium iodidestaining of cells were taken by flow cytometric analyses which confirmed no significant difference in membrane-compromised to membrane intact cells (data not shown). Proteins were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and searching sequences through database resulted in identification of approximately 60 proteins in A. baumannii supernatants (Table 1). A total of 23 proteins from different domain were identified from the band of SDS-PAGE. These proteins were mainly from protein Synthesis pathway, membrane Secreting protein, and efflux protein. A good number of proteins identified in SDS-PAGE were differentially secreted in ISR0031 (Table 1). Outer membrane proteins dominated the samples in both and overrepresented significantly in ISR0031, while few of efflux protein such as 33–36 kDa
2.3. Identification of protein bands The gel band were analyzed and those with visible clearly dense in either of control or test sample were excised for further process. In brief, the gel bands regions were excised after it was washed with ammonium bicarbonate (100 Mm) and treated with DTT (5 mM) to reduce the. Iodoacetamide (10 mM) was added following that it was washed 50 mM ammonium bicarbonate and dehydration with 100% acetonitrile. The gel pieces were completely dried, and digested with trypsin solution (Promega, Madison, WI) overnight at 37 °C. Peptides were removed using 60% acetonitrile/0.1% trifluoroacetic acid, and dried in vacuo. Samples were subjected to LC-MS analysis. 2.4. LC-MS-MS Analysis and Protein Identification Peptides were identified in a LTQ ion trap instrument (Thermofisher, LTQ) fortified with Surveyor HPLC pump (Thermofisher), Nanospray source, MicroAS autosampler and Xcalibur 2.0 SR2 control. Before running under LTQ-Ion trap sample were processed according to instruction and separated on liquid silicate similar to that previously described. The samples were run in to the injection to attain 700 nL flow-rates using mobile phase which contain 0.1% formic acid in acetonitrile at 400–2000 m/z with over nine scans (MS/MS). Tandem spectra were acquired and identity of proteins was searched through UniRef100 database using the SEQUEST algorithm. 2.5. Microarray analysis based on gene chips Overnight cultures of Isolate ISR0031 and A. baumannii ATCC adjusted to 0.5 OD in fresh LB medium then diluted up to two fold with ice-cold ethanol:acetone. Total RNA of sample was extracted following standard protocol earlier described (Janke et al., 2001). In brief, cells pellets were collected by centrifugation and disrupted by enzymatic lysis using lysozyme (10 mg/mL) in TE buffer containing proteinase K
Fig. 1. comparison between the supernatant protein of liquid culture of clinical isolates and ATCC strain. 2
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Table 1 The SDS-PAGE indicating the proteins which were differentially expressed in ISR0031, a clinically originated isolates. S. no
Description
ISR0031
ATCC
p-Value
Predicted MW (kDa)
Protein synthesis/chaperone proteins 1 Elongation factor 60 kDa chaperonin 2 Chaperone protein dnaK 3 Thiol:disulfide interchange protein
0.212 0.219 0.278
0.195 0.231 0.13866667
0.781 0.848 0.31433333
60.5 62.6 47
Membrane secreating protien 4 Outer membrane protein A 5 Putative outer membrane protein 6 Outer-membrane lipoproteins carrier protein 7 Peptidoglycan-associated lipoprotein 8 Lipoprotein
6.1705 2.997 3.0039 2.9716 0.5206
1.00375 0.7225 0.7467 0.3971 0.209
0.02025 0.04175 0.0798 0.0608 0.3629
38.4 37.05 25.091 20.82 29.88
Electron transport chain 9 ATP synthase subunit beta 10 ATP synthase subunit alpha
4.64196 0.11009
0.86355 0.1919
0.02525 0.02323
33.09 51.2
Bacterial programmed cell death 11 Bacteriocin (entericidin)
0.416
0.127
0.2
50.2
Cell wall biosynthesis 12 Murein lytic transglycosylase, soluble
0.685
0.48166667
0.32533333
53.76666667
Efflux protein 13 14 15 16 17 18
0.6095 0.3065 0.592 0.7015 1.23825 0.70725
2.859 0.1775 0.124 0.167 0.52575 0.2055
0.067 0.2095 0.0215 0.0385 0.1515 0.0725
35.35 66.65 42.15 37.7 29.025 20.625
Glucose metabolism 19 Enolase
2.01475
1.243
0.07875
38.875
Other 20 21 22 23
0.29304 1.20435 1.02342 0.16095
0.4551 0.71928 0.9546 0.25752
0.02775 0.78255 0.85914 0.33411
21.05 45.99 19.01 38.1
33–36 kDa outer membrane protein - associated beta-lactamase (AmpC) RND family drug transporter (AdeK) RND family drug transporter (AdeK) Putative signal peptide, metallo-beta-lactamase Putative signal peptide OsmY
Alkyl hydroperoxide reductase, C22 subunit Protein TolB precursor CsuA/B RecA
medical fraternity. A. baumannii sps have shown potential to represents the mounting burden of infections and also has proven challenging in the hospital due to its ability to persist (Queenan et al., 2012; Chan et al., 2007). Many a studies has established its virulence determinate using in-vitro approaches but those are yet to be verified in human model. This studies uses a proteomic approaches to identify the virulence factors and gene present in a clinical isolates ISR0031 of A. baumannii and compared their expression with A. baumannii strain ATCC 17978. The most important finding of these studies was 33–36 kDa outer membrane protein (omp), which is a porins having a major role in antibiotic transport through the outer membrane (CarO) was actively expressed in ATCC but lost the similar level of expression in ISR0031 which might be associated with resistance to carbapenems (Siroy et al., 2005). Study also observed the up-regulation of several genes associated with protein synthesis, membrane secreting protein and efflux protein. Few outer membrane proteins and gene associated with antibiotic resistance suggests that during the infection A. baumannii may have an impact on antibiotic resistance. Microarray analyses highlighted the differential expression of 36 gene representing include the membrane fusion AdeA efflux pump (AdeA) (Hood et al., n.d.; Magnet et al., 2001), which mediates resistance to a number of antibiotics in A. baumannii. Expression of this gene was shot up to 25 times folds than ATCC stain. Proteomic analyses highlighted an increased abundance of ABC transporter which is essential in cell viability, virulence, and pathogenicity. ABC trabsporters are supportive for iron ABC uptake systems, which are important effectors of virulence. The rise of extensively virulent A. baumannii is not witnessed so far but the growth in drug resistant capable organism in this species causes lethal infections upsurging a public health crisis particular challenge due to its adaptability within the hospital and host environments and the ability to readily
associated with membrane, signal peptide were major significant one. ATP synthase subunit beta was another major under electron transporter chain which was over expressed in clinical isolates. Proteomic analyses of supernatant proteins writes the details of release of proteins into culture media and determine if there is significant transcriptionally mediated changes in these two isolates. These was determined by microarray analyses using RNA isolated from stationary culture of bacteria and analyzed under hybridization of Affymetrix GeneChip® arrays. Total of 135 genes were found to be differentially regulated in ISR0031 (Table 2). Majority of genes were found to be involved in secondary metabolite biosynthesis, catabolism, transport and transcriptional regulation. There were seven major protein which were up regulated ten folds in ISR0031, as listed here-AdeA membrane fusion protein, putative ferric acinetobacter in transport system, Multidrug efflux transport protein, heavy metal RND efflux outer membrane protein (CzcC) putative ABC transporter, putative ABC-type antimicrobial peptide transport system, ABC-type nitrate/ sulfonate/bicarbonate transport systems. Interestingly, we observed down-regulation of AdeK proteins same time. (Table 2) and constitutive expression of OmpA, and AmpC, all of these were increased in culture supernatants of ISR0031. We also noticed that few proteins that increased in abundance in culture supernatants did not show much significant changes at transcript levels, indicating a possibility these proteins might be representing a post-translational mechanism for down regulation of target.
4. Discussion Acinetobacter baumannii is nosocomial pathogens and mainly upsurge pathogenesis in intensive care unit patients. A. baumannii is symbolic of the impending public health emergency threatening the 3
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5. Conclusions
Table 2 Proteomic approaches (microarray) analysis showed the genes differentially expressed in. S. no
Description
Fold-induction
p-Value
1 2
AdeA membrane fusion protein Putative ferric acinetobactin transport system Multidrug efflux transport protein Heavy metal RND efflux outer membrane protein (CzcC) Putative ABC transporter Putative ABC-type antimicrobial peptide transport system ABC-type nitrate/sulfonate/bicarbonate transport systems Outer membrane protein A ATP synthase subunit beta Transporter, LysE family Putative RND family cation/multidrug efflux pump Putative MFS transporter Outer-membrane lipoproteins carrier protein Peptidoglycan-associated lipoprotein Putative RND family drug transporter Major facilitator superfamily Heavy metal efflux pump (CzcA) Potassium-transporting ATPase A chain 33–36 kDa outer membrane protein associated Putative transporter DMT family permease Enolase Putative ATP-binding component of ABC transporter Putative signal peptide, metallo-betalactamase Protein TolB precursor CsuA/B Putative signal peptide OsmY RND family drug transporter (AdeK) Bacteriocin (entericidin) Beta-lactamase (AmpC) Alkyl hydroperoxide reductase, C22 subunit Thiol:disulfide interchange protein Chaperone protein dnaK Elongation factor 60 kDa chaperonin RecA ATP synthase subunit alpha
25.9 23.2
0.065975 0.137865
17.6 16
0.0211393 0.0018225
14.3 11.8
0.019565 0.77168
10.1
0.0020907
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
6.1705 4.64196 3.9 3.2
0.0716625 0.0229775 0.019565 0.0037575
3.2 3.0039
0.072618 0.7121205
2.9716 2.9 2.7 2.6 2.4 2.2585
0.7818174 0.07029 0.055328 0.02829 0.190645 0.067
2.2 2.1 2.01475 2
0.0379925 0.007182 0.07875 0.190645
1.23825
0.1515
1.20435 1.02342 0.70725 0.7015 0.416 0.3065 0.29304 0.278 0.219 0.212 0.16095 0.11009
0.78255 0.85914 0.0725 0.0385 0.2 0.2095 0.02775 0.065975 0.137865 0.035035 0.33411 0.02323
Up-regulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics and that is the major cause in A. baumannii explained how it poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane. Author contribution All authors contributed equally to the Literature review, Study plan, Lab work, fund generation, Manuscript writing, analyses data and proof. Declaration of competing interest The authors declare no conflict of interest. The authors themselves are responsible for the content and writing of the paper. Acknowledgement The authors would like to thank Mustansiriyah University (https:// uomustansiriyah.edu.iq/)Baghdad, Iraq for its support to complete this work. Ethical statements for human/animal experiments The study was approved by institutional ethics committee “University of Mustansiriyah” and informed consent was obtained in written by each individual participants. Each participant was known about the study follow up before enrolling for the study. I confirm that all experiments were performed in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and material All data available in manuscript. References
acquire resistance (Magnet et al., 2001; Marchand et al., 2004; Peleg et al., 2007). Similar to mammals, pathogens have evolved to acquire the defenses and proliferation results in strains that modify the host response to bring down pathogen clearance. The precise mechanism for attenuating such effect in this organism is not known. The results from these studies are very evident that a number of transporters like ABC, RND family efflux pump, Ade A and Ade K expression were differentially expressed in clinical isolates. These transporters have been investigate for their role in bacteria and its established fact that they have big role to play for bacterial resistant abilities (Pournaras et al., 2006; Al-Kadmy et al., 2018; Kareem et al., 2017; AL_Jubori et al., 2016). Multidrug efflux transporters were another important gene boosted in the clinical isolated almost 17 folds. This is a powerful mechanism extrude a number of structurally unrelated antibiotics classes from the bacterial cell, also includes toxic heavy metal ions, assisting their survival in harmful environments. Furthermore, upregulation of efflux pumps and down-regulation of outer membrane porins leads to rapid acquisition of resistance to new antibiotics (Peleg et al., 2007; Pournaras et al., 2006; Kareem et al., 2017; AL_Jubori et al., 2016). A. baumannii poses a particular challenge due to the intrinsic drug resistance imparted by its impermeable outer membrane.
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