Multidrug resistance behaviors of clinical Pseudomonas aeruginosa strains associated to pigment coloration

1Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, India; 2Department of Pharmacology, All India Institute of Medical Sciences, Rishikesh-249203, India; 3Department of Biochemistry, All India Institute of Medical Sciences, Rishikesh-249203, India; 4Department of Medical Oncology, All India Institute of Medical Sciences, Rishikesh-249203; 5Department of Cancer Biology, Central Drug Research Institute, Lucknow-226031.


Graphical Abstract:
Pseudomonas aeruginosa is an adaptable bacterial pathogen that infects various organs, including the respiratory tract, vascular system, urinary tract, and central nervous system leading to high morbidity and mortality. Our primary focus of this study was to characterize P. aeruginosa clinical strains on the basis of pigment color production, determine its association to multidrug resistance behavior and ability to form biofilm. We identified yellow and non-pigmented one (28.5%). Notably, green pigment producing strains when compared to non-pigmented groups also displayed antibiotic susceptibility behavior similar to yellow pigment producing strains. Although yellow pigment producing strains were strong biofilm producers, no significant association was identified between pigment and biofilm formation. Among pigmented and non-pigmented strains, majority of yellow pigment producing strains have shown MIC levels greater than the green and non-pigmented strains. Our study has demonstrated the impact of pigment coloration on susceptibility to antimicrobial agents where yellow pigment producing strains represent considerably a serious problem as due to lack of alternative agents against such transformed strain may collectively be associated with multidrug resistance development.

Introduction
Difficult to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) Pseudomonas aeruginosa strains have become the leading cause of nosocomial infection to human beings and pose life challenging threat in health care settings to immunocompromised patients [1][2][3][4]. Though limiting-dose of antibiotics given in bits may strengthen sensitive bacteria and transforme them into resistant version, the other imperative factors such as bacterial genomic mutations and acquisition of resistance genes particularly those encoding extended-spectrum β-lactamases (ESBL) or carbapenemases through horizontal gene transfer are also attributed to acquired resistance [5][6][7][8]. Notably, spread of so-called "high-risk" clones of P. aeruginosa poses a threat to global public health that requires extensive microbiological study and disease management to prevent their spread [8].
Pigments have functional roles in every organism, including lower organism such as microbes and higher ones, plants and animals. Microbial pigments can be primarily present in diffusible/water-soluble form where pigment is secreted out into the media and in non-diffusible form where pigment is retained within the organism. Most of the P. aeruginosa produces one or more extracellular pigments including pyoverdine (yellow-green and fluorescent), pyocyanin (blue-green), pyorubrin (red-brown), and pyomelanin (brown-black) [9,10]. These pigments are involved in multiple phenomenona such as quorum sensing network, virulence factor, antioxidant, and iron acquisition properties [11][12][13].
Functional implications of pigment production in P. aeruginosa pathogenesis have been acquainted with a number of studies. Laura et al have demonstrated contribution of pyomelanin production in P. aeruginosa provided resistance to oxidative stress and persistent chronic infection properties [13]. Notably, a strong correlation between pyoverdine production and virulence behavior of P. aeruginosa strain (isolated from cystic fibrosis patients) in a murine pneumonia model has been documented where disruption of pyoverdine production by specific inhibitor 5-fluorocytosine improved mice survival during infection and mitigated multidrug-resistant pathogenesis [14]. Pigment production appeared to be associated with virulent properties as expressions of virulence-associated genes (exoS, rhlA and rhlB) were more predominant in pigmented isolates than the non-pigmented clinical strains, suggesting that analyzing pigment production in clinical isolates can be a good initiative to detect virulence status of clinical strains [15].
Other important factors such as production of different class of enzymes called "β-lactamases" which include ESBLs, AmpC β-lactamases and metallo-β-lactamases (MBL) and non-enzymatic mechanism (overproduction of efflux pumps and outer membrane impermeability) are found to be associated with increased virulence and MDR behavior of P. aeruginosa isolated from clinical strains [16][17][18][19]. Notably, association of these factors with biofilm production which is another contributor of pathogenesis of P. aeruginosa has also been defined [20][21][22][23]. However, studies describing the clinical association between pigment production, MDR behavior and biofilm production are very limited [24,25]. In this study, we have presented association of pigment production status with MDR behavior, biofilm production, β-lactamases producing and efflux group of P. aeruginosa strains isolated from various clinical isolates at All India Institute of Medical A total of 143 consecutive samples were selected of those identified to be infected with P.
aeruginosa strain. Samples from various sites including pus, pleural fluid, urine, blood, sputum, and other discharges were collected from infected patients enrolled in respective departments, outside patients (OPD), inside patients (IPD) and intensive care unit (ICU). Samples after collection were immediately transported to the Microbiology lab and processed immediately as per routine hospital procedure of examination including bacterial identification, gram staining (HIMEDIA, K001), colony morphology (size, shape, texture, opacity), motility (hanging drop method), pigment production (identified by color production), oxidase reaction (Oxidase disc, DD018), and other routine microbiological procedures [26]. Further species identification for the isolates was carried out using Bruker's MALDI Biotyper Microbial Identification system (Bruker, USA).

Pigment identification
Color of pigment production was detected by qualitative observation of bacterial growth on preformed King's A medium agar (HiMedia, M1543). Inoculated bacteria were allowed to grow for 16 hours at 37°C. Colonies those appeared blue-green in texture were considered pyocyanin producers, while-yellow colonies were considered as pyoverdine producers (Supplementary figure 1A).

Antibiotic susceptibility test (AST)
We have performed a disc diffusion method to test the AST of P. aeruginosa strains according to the Kirby-Bauer disk diffusion method on Muller-Hinton agar (MHA) [27]. We have also utilized an automated method for AST detection using a MicroScan WalkAway 96 Plus ID

Extended-spectrum β-lactamase detection.
ESBL production from all P. aeruginosa isolates was detected by Beckman Coulter Microscan Walk-Away and double-disc synergy methods. ESBL production was screened by disc diffusion assay using ceftazidime (30μg) and ceftazidime/clavulanic acid (30/10μg) discs and Piperacillin (100μg) and Piperacillin/tazobactam (100/10μg) discs. The zones of inhibition for the ceftazidime and Piperacillin discs were compared to ceftazidime/clavulanic acid and Piperacillin/tazobactam discs. An increase in zone diameter in presence of tazobactam was confirmed as positive for ESBL production according to CLSI 2021 guideline. ESBL positive ATCC 27853 was used as a control strain. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug-resistant [28].

Minimal inhibitory concentration (MIC) of MBL producing P. aeruginosa
To detect MBL production by P. aeruginosa isolates, we performed RAPIDEC® CARBA NP test (Biomerieux-diagnostic) that is rapid and well adapted to detect carbapenemase producers as manufacturer instructions and as described previously [29]. A positive test for MBL producers corresponded to a color change from red to yellow or orange or light orange or dark orange. No color change indicated MBL-non producers strains (Supplementary Figure 2 2.6 AmpC β-lactamases production phenotypic detection.

Biofilm assay and quantification
Biofilm formation assay was performed as described previously [30]. In brief, overnight culture of P. aeruginosa strain was diluted to OD of 0. 5

Statistical analysis
All data were analyzed and plotted using GraphPad Prism 5.02 software (GraphPad, La Jolla, CA, USA) and Microsoft excel. Differences between the groups and statistical significance were determined using Chi-square test. P < 0.05 was considered statistically significant.

Prevalence of pigmented and non-pigmented P. aeruginosa clinical strains
A total of 143 clinical isolates were obtained from OPD, IPD and ICU departments that were identified to be infected with P. aeruginosa. Among them, 92 (64.5%) appeared in IPD department. Of the 143 patients, 93 (65.1%) were males and 50 (34.9%) were females (P < 0.05).
Determination of mucoid and non-mucoid isolates was investigated phenotypically based on colony appearance and Congo red agar assay. Both non-mucoid 66 (46.1%) and mucoid 77 (53.8%) groups of P. aeruginosa were identified (P > 0.05) ( Table 1). Based on pigment production in Kings medium, 43 (30.1%) of 143 archived isolates produced yellow pigment, whereas 57 (39.8%) were green and 43 (30.1%) with no pigment producing strains respectively (P > 0.05) ( Figure 1A). As number of samples collected from IPD department was higher, therefore the relative distribution of green, yellow and no pigment producing strains were higher in IPD then the other hospital department ( Figure 1B).

Antimicrobial resistance pattern of pigmented and non-pigmented P. aeruginosa clinical strains
We next evaluated the antibiotic susceptibility pattern using a group of 14 anti-pseudomonal drugs. Pigmented strains (green and yellow producing strains) showed profound resistance to tested antibiotics compared to non-pigmented strains ( Figure 1C). Among the pigmented and non-pigmented groups, the yellow pigment producing strains were more resistant to majorly of drugs tested then green and no pigment producing strains ( Figure 1C and Supplementary Figure   1B, C  Figure 1C). Similar results were obtained when patients were grouped into OPD, IPD, and ICU department, where yellow pigment producing strains presented a more frequent resistant pattern then green and non-pigmented strains ( Figure 1D). Notably, patients from ICU showed profound resistance patterns in yellow pigment producing strains then IPD and OPD patients ( Figure 1D and Table 2).
On distribution of antibiotics tested into different classes including aminoglycosides carbapenems, β-lactam and others (fluoroquinolones, polymyxin, sulfonamides), we found majority of P. aeruginosa strains were resistant to β-lactam class of antibiotics ( Figure 2A and Table 2). Based on pigment production, pigmented strains were more frequently resistant than the non-pigmented strains. We found yellow pigmented strains were strongly and significantly resistant to all classes of antibiotics followed by green and non-pigmented strains ( Figure 2B-E).
We further evaluated the strains that were positive producers of ESBL, MBL, AmpC and showed efflux activity. We determine frequency of strains as single enzyme or efflux positive producers and co-producers strains (positive for two or more than two enzymes or enzyme plus efflux). The yellow and green pigment producing strains were equally distributed (46.6%) among single enzyme/efflux positive producers, whereas non-pigmented strains were less frequent (6.6%) ( Figure 3C). Where, yellow pigment producing strains were highly frequent (61.9%), then green (19.04%) and non-pigmented (19.04%) strains among co-producers strains ( Figure 3D).

Biofilm production of pigmented and non-pigmented strains
Mucoid and non-mucoid colonies are associated with biofilm production and resistance to antibiotics respectively. We do not observe significant difference in biofilm formation between mucoid and non-mucoid strains (Supplementary Figure 4A). However, mucoid strains were frequently resistant to piperacillin/Tz, piperacillin, meropenem, aztreonam and doripenem then non-mucoid strains (Supplementary Figure 4B). Among the mucoid and non-mucoid groups there was no relative difference observed in frequency of yellow, green and non-pigmented strains ( Figure 4A). To test biofilm formation ability of clinical strains, we screened all 143 yellow, green and non-pigmented P. aeruginosa strains. We did not find correlation of antibiotic resistance pattern between biofilm and non-biofilm producers (Supplementary Figure 4C). Out of 143 strains, 106 (74.1%) were biofilm producers, in which 34 (32.1%) were yellow, 46 (43.4%) green and 26 (24.5%) non pigmented strains produced biofilm ( Figure 4B). Based on OD received on microtiter plate, we have further categorized biofilm producers as strong, moderate and weak biofilm producers. Comparative analysis among yellow, green and non-pigmented strains showed 16 (37.2%) of yellow, 17 (29.8%) of green and 9 (20.9%) of non-pigmented strains were strong biofilm producers. Whereas 6 (13.9%) of yellow, 13 (22.8%) of green and 12 (27.9%) of non-pigmented were moderate biofilm producers; and 6 (27.9%) of yellow, 13 (28.1%) of green and 12 (11.6%) of non-pigmented were weak biofilm producers ( Figure 4C).
3.5 MIC of MBL producing pigmented and non-pigmented strains.
MBL producing P. aeruginosa strains are a threat to individuals and are associated with higher morbidity mortality rate especially in immunocompromised patients [31]

Discussion
The increasing incidence of MDR bacterial strains particularly for most common clinical pathogen P. aeruginosa in human isolates poses a significant challenge to identify new treatment strategies that can lead to high morbidity and mortality in hospitals. Fortunately, a number of rapid methods have been developed to identify such MDR species along with antibiotic susceptibility within a couple of hours from clinical isolates that have benefited to reduce increasing mortality [32][33][34][35].
Common characteristics for P. aeruginosa to represent the MDR behavior in clinical strains are presence of drug resistance genes or its mutant variety in bacterial plasmid or in its genome, high expression of β-lactamase group of enzymes, and up regulation of drug efflux pump have been documented [36][37][38][39]. Interestingly, a recent effort using proteomic profiling has characterized antibiotics sensitive and MDR clinical strains of P. aeruginosa that provided non-genetic changes associated with antibiotic susceptibility responses [40].
Pigment production such as pyocyanin and pyoverdin are important virulence factors that augment bacterial virulence via diverse mechanisms [13][14][15]. According to literature search and best of our knowledge, characterization of MDR signatures of P. aeruginosa from clinical strains on the basis of pigment production are very limited [13][14][15]. In this study, we have presented a strong association of pigment coloration produced by P. aeruginosa strains from clinical isolates with MDR behavior, efflux activity and biofilm formation. Primarily, we notified three major groups of P. aeruginosa: green, yellow and no pigment producing strains from our cohort of clinical samples. Note: we have also identified intermittent red and brown pigment producing strains, and we lacked their adequate numbers for analysis, therefore, we have excluded them from this study.
Among the pigmented and non-pigmented strains, yellow pigment producing strain showed profound resistance behavior with majority of antibiotics tested in clinical samples archived from IPD and ICU department, suggesting that this yellow pigment producing strain can be a serious risk factor. Remarkable resistance pattern of yellow pigmented strain was noted for different classes of antibiotics groups including aminoglycosides, fluoroquinolones, carbapenem, sulfonamides, polymyxin and β-lactams.
Acquired resistance by the production of ESBL, MBL and AmpC enzymes is a common phenomenon P. aeruginosa [41]. Phenotypic methods applied in this study helped in detecting P.
aeruginosa isolates producing various ESBL, MBL, AmpC, enzymes, and efflux activity against different antibiotics. Though, frequency of these enzymes producing strains from our cohort of 143 clinical isolates was not higher as shown by other studies, the prevalence of such producing organisms could depend on geographic origins, infection patterns, hospital infection control measures, and different departments within the same hospital [16,35]. Notably, our yellow pigment producing strains were more frequent enzyme producers and displayed co-occurrence with more than one enzyme/efflux positivity than green and non-pigmented strains. In addition, identification of MIC for MBL producing strains presented an augmented MIC level of yellow pigment producing strain than the CLSI MIC breakpoints.
Biofilm formation is an important mechanism P. aeruginosa survival causes considerable problems and these structures provoke greater resistance to the treatment with antibiotics.
Inverse correlation between biofilm formation with expression of MDR genes has been identified in few studies [42,43]. In our study, though a significant number (74.13%) of isolates formed biofilm, we did not observe a strong correlation of biofilm formation with pigment production.
Conclusively, green pigmented strains showed moderate resistance pattern when compared to yellow and non-pigmented strains suggesting that pigment producing strains could be associated to resistance to antimicrobial agents then the non-pigmented ones and requires comprehensive testing for antibiotics susceptibility pattern and to prevent detrimental effects before a treatment recommendation.

Conclusion
Our study has presented an association of pigment coloration with MDR behavior of P. aeruginosa isolated from various sources of clinical samples. Moreover, this study demonstrated ESBL, MBL, AmpC and Efflux mediated resistance among the different pigmented P. aeruginosa isolates.
Though we have not identified the associated gene expression or genetic mutation or other mechanism connecting pigment production with MDR, which could be a further area of future research. We found yellow pigment color producing P. aeruginosa strains displayed resistant patterns to more than one type of antibiotic groups. Therefore, identification of right choice of antibiotic for treatment is critically important as misuse or overuse of antibiotics can cause significant risk of emergence of antibiotic resistance. Since pigment production is easy to determine, this might be a good starting point to identify the multi-drug resistance status of an isolate. However, further study is required to confirm this observation.

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