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Article

Insights into the Antibacterial Activity of Prolactin-Inducible Protein against the Standard and Environmental MDR Bacterial Strains

by
Mohd Yousuf
1,
Asghar Ali
1,
Parvez Khan
2,
Farah Anjum
3,
Abdelbaset Mohamed Elasbali
4,
Asimul Islam
2,
Dharmendra Kumar Yadav
5,*,
Alaa Shafie
3,
Qazi Mohd. Rizwanul Haque
1 and
Md. Imtaiyaz Hassan
2,*
1
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
2
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
3
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
4
Department of Clinical Laboratory Science, College of Applied Medical Sciences-Qurayyat, Jouf University, Sakakah 42421, Saudi Arabia
5
College of Pharmacy, Gachon University of Medicine and Science, Hambakmoeiro 191, Yeonsu-gu, Incheon City 21924, Korea
*
Authors to whom correspondence should be addressed.
Microorganisms 2022, 10(3), 597; https://doi.org/10.3390/microorganisms10030597
Submission received: 25 February 2022 / Revised: 4 March 2022 / Accepted: 8 March 2022 / Published: 9 March 2022
(This article belongs to the Special Issue State-of-the-Art Clinical Microbiology Technology in Korea)
Browse Figures
Versions Notes

Abstract

:
Background: Prolactin inducible protein (PIP) is a small secretary glycoprotein present in most biological fluids and contributes to various cellular functions, including cell growth, fertility, antitumor, and antifungal activities. Objectives: The present study evaluated the antibacterial activities of recombinant PIP against multiple broad-spectrum MDR bacterial strains. Methods: The PIP gene was cloned, expressed and purified using affinity chromatography. Disk diffusion, broth microdilution, and growth kinetic assays were used to determine the antibacterial activities of PIP. Results: Disk diffusion assay showed that PIP has a minimum and maximum zone of inhibition against E. coli and P. aeruginosa, respectively, compared to the reference drug ampicillin. Furthermore, growth kinetics studies also suggested that PIP significantly inhibited the growth of E. coli and P. aeruginosa. The minimum inhibitory concentration of PIP was 32 µg/mL for E. coli (443), a standard bacterial strain, and 64 µg/mL for Bacillus sp. (LG1), an environmental multidrug-resistant (MDR) strain. The synergistic studies of PIP with ampicillin showed better efficacies towards selected bacterial strains having MDR properties. Conclusion: Our findings suggest that PIP has a broad range of antibacterial activities with important implications in alleviating MDR problems.

1. Introduction

Innate immunity plays an important role in human health [1]. During infection, injury, and disease conditions, the metabolism or nutritional homeostasis of the body is disturbed, involving various signaling pathways and secondary messenger molecules [2,3]. Innate immune responses protect our body through various alterations such as modulating membrane composition, antioxidants or cellular redox levels, and cytokines production [4,5]. In addition, the innate immune system provides the first line of defense mechanism against different types of bacteria by secreting multiple types of defense proteins and peptide molecules such as lysozyme, secretary leucoprotease, and lactoferrins that kill or inhibit bacterial growth [6]. The effector molecules are low-molecular-weight peptides and work as multifunctional molecules; one such small protein is a prolactin-inducible protein (PIP), found in different body secretions, where it controls bacterial growth and binds to several other partner molecules to exhibit multiple functions in cancer growth, fertility, and enamel pellicle formation [7,8,9,10,11,12,13].
PIP plays an important function in immune defense and the reproductive system, and its expression is upregulated by prolactin or androgens, whereas it is downregulated by estrogens [14,15]. During pathological condition like corneal disease, keratoconous, dacryliths, and cancer, the expression of PIP get altered in several exocrine tissues or mammary glands such as sweat and salivary glands, and thus acting as a biomarker in such diseases [16,17,18,19]. PIP is a small single polypeptide chain protein expressed in various human body parts, including the salivary gland, lacrimal gland, trachea, prostate, muscle, mammary glands, and lungs. The highest expression of PIP was reported in the salivary gland (~55%) [16,20]. The gene of human PIP has located on chromosome 7q32-36 regions, encoding for a 146 amino-acid-residue-long polypeptide leading to the synthesis of a ~17 kDa protein [16,21,22].
PIP is a β-rich glycosylated protein. The crystal structure of PIP demonstrated that β sheets are organized around the hydrophobic amino acid residue and form a sandwich-like structure. PIP contains Asn-X-Ser/Thr motif present at position Asn77-Arg78-Thr79, representing a potential glycosylation site [21]. The structural domain of PIP represents immunoglobulin domains [23,24]. Previous studies on the structure and function of PIP demonstrated its isolation from different biological fluids, where it performs numerous functions with its interacting partner proteins [16,25,26,27]. The presence of immunoglobin domains suggested PIP’s immunological implications supported by multiple studies using in vitro and in vivo (mouse models) presenting the role of PIP in innate and cell-mediated immunity [28,29].
The expression of PIP is used as a marker of mammary cell differentiation, and its altered expression is associated with breast cancer progression [29]. Recent studies suggested that the downregulation of PIP promotes osteogenic differentiation of periodontal stem cells [30]. Multiple reports supported the antimicrobial or anti-protozoal roles of PIP. They suggested that reduced levels of antimicrobial or immunomodulatory proteins in tears lead to higher ocular infections and susceptibility to other pathogens such as Leismania major [31,32]. PIP acts on oral bacteria (an oral defense mechanism), promoting its clearance from the oral cavity and modulating oral flora [33]. PIP is present in most of the biological/physiological fluids of the human body. However, the antibacterial function of PIP is not well-studied and little work has been conducted on evaluating its antibacterial properties. Thus, we aimed to investigate the antibacterial activity of PIP on multiple microbial strains, including MDR strains.

2. Materials and Methods

2.1. Bacterial Strains and Plasmids

The coding region of the PIP gene was amplified from total human cDNA through PCR using PIP-specific primers bearing NdeI (forward primer) and XhoI (reverse primer) restriction sites. The amplified amplicon was digested with NdeI and XhoI, and subsequently cloned into pET28a+ (Novagen, Madison, WI, USA) prokaryotic expression vector. DH5α and BL21 (DE3) strains of E. coli cells were used for PIP cloning and expression, respectively. Plasmid isolation, restriction enzyme digestion by NdeI and XhoI, and the ligation experiments were performed as described previously [34]. Luria Broth (DifcoTM, Becton Dickinson, Fisher Scientific, Kansas City, KS, USA) was used for bacterial culture with 50 µg/mL Kanamycin (Sigma, Saint Louis, MO, USA).

2.2. Expression and Purification of PIP

The pET28a+-PIP expression construct was transformed in E. coli BL21 (DE3) following the standard protein expression protocol as described previously [35]. The primary culture grown overnight was used to develop secondary culture containing 50 µg/mL kanamycin and incubated at 37 °C with constant agitation at 180 rpm in an incubator shaker until the absorbance reached 0.6 at 600 nm. The secondary culture was induced by 0.5 M IPTG (Sigma, Saint Louis, MO, USA) and incubated for an additional 4–5 h at 37 °C, 180 rpm.
The cell culture was centrifuged at 7000–8000× g for 10 min, and the cell pellet was dissolved in cell lysis buffer having 50 mM Tris–HCl buffer, pH 8.0, 200 mM NaCl, 2% (v/v) glycerol, 1 mM β-mercaptoethanol, 0.1 mg/mL lysozyme, 1 mM phenyl methane sulfonyl fluoride (PMSF), and 1% (v/v) triton X-100 (U. S. Biochemical Corp, Cleveland, OH, USA) and incubated for 1 h at 37 °C. Following the incubation, cell lysate was sonicated on ice for 15 min and centrifuged for 30 min at 13,000 rpm at 4 °C. The supernatant was collected, and protein expression was checked using SDS-PAGE. Ni-NTA affinity chromatography was used for the purification of 6XHis-tag-PIP protein. For this, the clear supernatant was passed through Ni–NTA column pre-equilibrated with Tris buffer (50 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1% v/v glycerol, 20 mM imidazole). Following protein binding, the column was washed with 50 mL of washing buffer (50 mM Tris–HCl, pH 8.0, 200 mM NaCl, 50 mM imidazole) at 4 °C. Bound protein was eluted with an imidazole gradient (100–300 mM). The eluted fractions were run on the SDS-PAGE to check the purity of the protein. Fractions containing purified protein were pooled and dialyzed in 20 mM Tris–HCl buffer, pH 8.0, 100 mM NaCl, and 2% glycerol. The dialyzed protein was concentrated using Amicon Ultra 5 K device (Merck, Darmstadt, Germany). The concentrated protein sample was further loaded on Hi Trap DEAE-FF (1 mL, 7 mm × 25 mm) column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with 50 mM Tris–HCl buffer, pH 8.0. Bound proteins were eluted with increasing concentration of NaCl (0–1 M) in the 50 mM Tris–HCl buffer, pH 8.0. PIP eluted at 0.50 M NaCl was pooled, concentrated, and stored for further studies. The purity of PIP was tested by SDS-PAGE and validated through Western blot using the luminol method [36].

2.3. Antibacterial Activity Assays

Subculturing of standard bacterial strains, including S. aureus (MTCC 902), B. subtilis (MTCC 736), P. aeruginosa (MTCC 2453), Escherichia coli (MTCC 443), and environmentally resistant (multidrug-resistant) bacterial isolates, i.e., Citrobacter werkmanii (SH 52/MN267555), E. coli (SD6/MT577556), Bacillus sp. (LG1/MT576690), and Citrtobacter sp. (HK 106/MT576965) was performed on nutrient agar medium through the streaking method [37]. Following the streaking, the plates were kept in the incubator overnight at 37 °C, and the growth of each plate was observed on the next day.

2.4. Determination of MIC and MBC

To determine the MIC of the PIP, the microdilution method was performed according to the standard protocol of NCCL [38]. MIC of the PIP was determined against two Gram-positive (B. subtilis and S. aureus) and two Gram-negative (P. aeruginosa and E. coli) bacterial strains. The MIC values were estimated and compared to ampicillin (AMP), used as a control antibiotic. To obtain the stock solution of 10.24 mg/mL for PIP and AMP (standard bacterial drugs), they were dissolved in Tris buffer and sterile water, respectively. The serial dilutions of broth were performed to obtain the final concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL. A control test with buffer was performed following the same dilutions to check whether the buffer or solvent of PIP affected the growth of bacteria. The cultures were incubated at 37 °C for 24 h and compared with blank in terms of turbidity developed by the microbial growth. The minimum concentration of PIP exhibiting no bacterial growth was described as MIC. For the MBC, 10 μL aliquots from each well that showed no growth of microorganism were plated on Mueller–Hinton Agar (MHA) and incubated at 37 °C for 24 h [39,40]. The MIC and MBC values were calculated as per the previously published protocols [41] and compared with the AMP taken as a reference antibiotic.

2.5. Disk Diffusion Assay

Disk diffusion assay was performed to identify the antibacterial efficacy of PIP against standard bacterial strains by following Kirby–Bauer method [42]. Various concentrations of PIP such as MIC/2, MIC and 2MIC were taken on disc for disc diffusion assay [43,44,45,46]. Briefly, the bacterial cells were inoculated in a liquid broth medium and grown overnight at 37 °C. Approximately 105 cells/mL were taken from that liquid broth medium, inoculated into molten nutrient agar medium, and poured into Petri plates [45,47,48,49]. After solidification, autoclaved Whatman papers having 4 mm diameter disks were put at suitable distances over solid agar, and MIC/2, MIC, and 2MIC from the stock solution were put on disks. Plates were incubated at 37 °C overnight, and the next day, ZOI was measured in mm.

2.6. Combination Studies of PIP against the Standard Bacterial Strains

The synergistic activity of PIP with standard drug AMP was determined using the microdilution checkerboard method against standard bacterial strains of E. coli and P. aeruginosa as described previously [50]. AMP were serially diluted in columns from 32,16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/mL, while PIP was diluted in rows from 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1.0, and 0.5 μg/mL, respectively, in a 96-microwell plate to obtain multiple combination. The plates were inoculated with a freshly prepared culture of bacterial isolates and incubated at 37 °C overnight. Following the incubation time, combinatorial MIC was determined as the concentrations at which no visible growth occurred. Using the formula given below, the synergy of compounds in terms of FICI (fractional inhibitory concentration index) was calculated [41]:
ΣFIC = FICA + FICB = (CA/MICA) + (CB/MICB)
where MICA and MICB are the MICs of drugs A and B alone, respectively, and CA and CB are the concentrations of the drugs in combination, respectively, in all of the wells corresponding to an MIC (isoeffective combinations).
Synergy and antagonism were defined by FICI indices ≤ 0.5 and >4, respectively, and ‘indifferent’ was defined by 1 < FICI ≤4.

2.7. Growth Kinetics Assay

Based on previous experiments, we have selected E. coli and P. aeruginosa for these experiments. Bacterial cells were freshly revived by sub-culturing on the Luria agar plate. To obtain the fresh cultures for the experiments, an inoculum was transferred into the Luria broth and was grown overnight at 37 °C. Different concentrations of PIP, equivalent to MIC/2, MIC, and 2MIC, were added separately to the respective conical flasks containing inoculated medium and incubated at 37 °C. Positive control was also taken to observe the full growth. To observe the growth kinetics of the cultures, 1 mL aliquot of each sample was taken out from the culture flask and growth was measured at 590 nm turbidometrically (optical density) using Thermo Multiskan spectrophotometer after each one-hour interval [51]. To determine the effect of PIP on bacterial growth, a graph was plotted between OD versus time duration (hours) to obtain a growth curve and further analysis [52].

2.8. Effect of PIP on Environmental Resistant Bacterial Strains

Antibacterial activity of PIP was investigated against a variety of environmental resistant strains such as HK 106 (Citrobacter sp.), LG 1 (Bacillus sp.), SD 6 (Escherichia coli), and SH52 (Citrobacter werkmanii). All bacterial strains were isolated from different environmental (Lake, Pond, effluent from slaughterhouse and wastewater treatment plant) water samples. The standard broth dilution method was used to determine the MIC of these isolates and compare the MIC values to the conventional antibiotics such as ampicillin (a penicillin analog). The microdilution checkerboard method has been used to examine the synergistic activity of the PIP with the standard antibiotic AMP against Escherichia coli and Citrtobacter werkmanii. The procedure above was used for the MIC and synergistic activity [41,53,54,55].

3. Result and Discussion

3.1. Cloning, Expression, and Purification of PIP

The PIP gene (from plasmid pcDNA) was amplified using a gene-specific PCR, with Ndel and XhoI sites in the forward and reverse primers, respectively. The size of the amplified product was 366-bp (Figure 1A). The amplified gene product having selected restriction sites was digested with NcoI and XhoI and subsequently ligated into the pET28a+ backbone digested with similar restriction enzymes (Figure 1B). The ligated product was transformed into E. coli and positive colonies were selected and confirmed using colony PCR. The positive clones were further confirmed using restriction digestion through NcoI and XhoI endonucleases (Figure 1C). Finally, the constructed plasmid was verified by DNA sequencing (Figure S1).
The confirmed plasmid construct pET28a+ with PIP was transformed into BL21 (DE3) strain of E. coli for protein expression. The recombinant protein expression was performed at 37 °C by inducing the bacterial cultures with 0.5 mM IPTG for 4–5 h. The over-expression of PIP was confirmed through SDS-PAGE, showing a prominent protein band corresponding to PIP with an apparent molecular mass of ~14 kDa (Figure 1D). The purification of PIP follows two-step processes. In the first step, PIP was purified using Ni-NTA chromatography (Figure 1E), followed by the second step of purification using ion-exchange chromatography (Figure 1F,G). A single band corresponding to the size of PIP was observed on the SDS-PAGE, confirming the purity of PIP (Figure 1G).

3.2. Determination of MIC and MBC

Multiple studies reported the presence of PIP in several body fluids, showing antibacterial activities [7,56,57]. To study the antibacterial activities of PIP, in particular, we have successfully cloned, expressed, and purified the PIP using a prokaryotic expression system. It is important to mention that the recombinant protein isolated from the prokaryotic expression system is devoid of post-translational modifications. We previously reported the crystal structure of PIP isolated from seminal human plasma. We noticed comparable structural properties of recombinant PIP, thus suggesting that recombinant PIP might be as active as the protein purified from body fluids [22,23].
Initially, the antibacterial activity of PIP was evaluated through the zone of inhibition (ZOI) measurements in diameter (mm) on MHA plates and serial dilution of broth to observe the growth of bacteria by measuring the turbidity of cultures. The estimated MIC value for the PIP against B. Subtilis and P. aeruginosa was 64 µg/mL, wh. In contrast, for E. coli and S. aureus the MIC values were 32 µg/mL and 128 µg/mL, respectively (Table 1). AMP was used as a reference antibiotic (Positive control). The results suggested that PIP has the highest inhibitory effect against E. coli among the tested strains. However, the MIC value for S. aureus bacterial strain appears to be comparatively high. Interestingly, PIP showed a similar profile activity (MIC values) towards B. subtilis and P. aeruginosa strains.
In addition, the MBC values of PIP range from 128 to 512 µg/mL (Table 1). The ratio of MBC to MIC is used to characterize the antibacterial activity of any compound. If the MBC/MIC ratio was ≤2, the compounds were termed bactericidal, and they were termed bacteriostatic if the ratio was between 2 and 16 [43,45,58,59,60,61]. The results shown in Table 1 suggested that the PIP showed bactericidal and bacteriostatic effects based on the tested bacterial strains.

3.3. Disk Diffusion Assay

The disc diffusion test is a widely accepted and primary test to see the susceptibility of bacteria or bacterial isolates towards antibiotics or any antibacterial molecules [46,49,62,63]. To further evaluate the antibacterial activity of PIP, following the serial dilution method, the disk diffusion assay was used. The disk diffusion experiment results revealed that PIP showed the lowest and highest ZOI against P. aeruginosa and E. coli, respectively (Figure 2). In Gram-negative E. coli culture, the treatment with PIP leads to a clear ZOI of 16, 20, and 26 mm around the disc of MIC/2, MIC, and 2MIC, respectively. While in the case of P. aeruginosa (2453), 6, 8, and 13 mm clear ZOI 2343 measured around the disks of the MIC/2, MIC, and 2MIC concentrations, respectively (Figure 2, Table 2). Around the disks of MIC/2, MIC, and 2MIC, the observed ZOI was 8, 9, and 12mm with B. subtilis and 8, 13, and 17 with S aureus, respectively. Antibacterial activities of PIP using agar diffusion assay are presented in Figure 2 and Figure 3. Diffusion assay results in terms of ZOI diameter (mm) for standard bacterial strains are given in Table 2.

3.4. Synergistic Antibacterial Activity of PIP

The development of multidrug resistance (MDR) in bacteria is a leading cause of bacterial-infection-related deaths [64,65,66,67,68,69]. Therefore, the development of combinatorial therapeutic strategies or synergistic approaches offered an effective therapeutic regimen to deal with MDR problems [64,70,71,72,73]. To investigate the synergistic effect of PIP towards the antibacterial activity of the standard drug (ampicillin), PIP was further evaluated in combination with AMP against selected standard bacterial strains. Similar synergistic experiments were also performed with environment MDR bacterial strains. Interestingly, the results of synergistic studies showed that PIP with AMP significantly enhanced the antibacterial activity of standard drugs. The results of synergistic studies are summarized in Table 3.
The results showed a substantial increment in the antibacterial activity of PIP against E. coli and P. aeruginosa strains when used in combination with AMP. The resistance pattern of all these bacterial isolates toward various antibiotics is described in Table 4. The outcomes of synergistic studies suggested the potential implications of PIP towards the development of future combinatorial therapeutic strategies to alleviate antimicrobial resistance.

3.5. Growth Kinetics

The kinetic growth studies provide an important tool to see the influence of substrate concentration or a molecule towards the specific growth rate (versus time) and provide a method to evaluate drug susceptibility to microbial growth [74,75]. Therefore, growth kinetics studies were performed to determine the effect of PIP on the growth of E. coli and P. aeruginosa. For this, we take untreated cultures as a negative control, and AMP-treated culture was taken as a positive control. The results showed that the growth curve of untreated bacterial cells (control) follows the standard growth curve with a clear lag, exponential or log, brief stationery, and decline phases of the bacterial cell. On the other hand, at 2MIC, MIC, and MIC/2 concentration values of PIP, no growth was observed until 20 h of incubation in E. coli growth experiments (Figure 4). In the case of P. aeruginosa at 2MICconcentration values of PIP, no growth was observed until 24 h. At MIC concentration values, a slight increase in the culture growth was observed after 20 h.
In contrast, at the MIC/2 concentration, the lag phase was increased from 3 to 8 h, the log phase was significantly increased from 9 h to 19 h, and following the log phase, a short stationary phase was observed from 20 h to 22 h, then a decline phase was observed. Moreover, PIP completely inhibited the growth of both bacterial strains at MIC and 2MIC concentrations. No growth was observed in AMP-treated bacterial cells for 24 h in both selected bacterial strains.

3.6. Effect of PIP on Environmental MDR Strains

The MIC value for the PIP against environmental MDR isolates was observed from 64–512 µg/mL. Minimum MIC values 64 µg/mL for LG1 64, whereas maximum MIC values 512 µg/ml for SD6 were observed among tested MDR isolates (as shown in Table 1). Environmental MDR isolates SH52, LG1, SD6 showed a significant decrease as 4, 3, 2-fold MIC values compared to AMP, respectively, while HK106 showed nearly the same MIC value for PIP and AMP (Table 1). MBC values ranges for the resistant strain varies from > 1024 to 512 µg/mL. If the MBC/MIC ratio was 2 to 8, and the compounds were termed bactericidal and bacteriostatic. The PIP has various bactericidal and bacteriostatic effects on environment-resistant bacterial isolates. The zone of inhibition assays for PIP against environmental-resistant bacterial isolate LG1 shows a maximum ZOI of 8mm at MIC/2 concentration and of 12 mm at MIC concentration. Moreover, at 2MIC concentration, the maximum ZOI was 14 mm. At the same time, Citrobacter werkmanii (SH 52) shows a ZOI of 7 mm at the MIC/2 concentration, 9 mm at the MIC concentration, and 12 mm at the 2MIC concentration. Furthermore, the combinational (PIP and AMP) studies against SD6 and SH52 showed that the PIP has a differential mode of interaction (FICI = 1.5) with AMP against SD6. In contrast, it showed the synergistic mode of interaction (FICI = 0.0097) against SH52 (Table 3).

4. Conclusions

To study the antibacterial potential of PIP, we cloned, expressed, and purified PIP using a prokaryotic expression system. The antibacterial activities of PIP were evaluated against a broad spectrum of bacterial strains. The outcomes of the present study suggest that PIP significantly inhibited a wide range of bacterial strains, including standard and environmental MDR strains. The enhanced antibacterial potential of standard antibiotics such as ampicillin in synergistic studies of PIP makes it an important investigatory molecule towards developing combinatorial antibacterial approaches to deal with the rising burden of antimicrobial resistance. This study indicates PIP’s future investigations as a novel bioactive molecule for developing antibacterial therapies and as a preservative agent in the food, cosmetic, and medicine industries.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms10030597/s1. Figure S1: Sequence of PIP gene.

Author Contributions

Conceptualization, M.Y. and P.K.; methodology, M.Y.; software, A.A.; validation, M.Y. and A.A.; formal analysis, F.A., A.I.; investigation, A.S.; resources, A.M.E.; data curation, Q.M.R.H.; writing—original draft preparation, A.A., M.Y.; writing—review and editing, A.I., P.K.; visualization, M.I.H.; supervision, M.I.H.; project administration, D.K.Y.; funding acquisition, M.I.H. and D.K.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Taif University Researchers Supporting Project Number (TURSP-2020/131), Taif University, Taif, Saudi Arabia, and the Indian Council of Medical Research (Grant No. ECD/Adhoc/2/2021-22).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This work was supported by Taif University Researchers Supporting Project Number (TURSP-2020/131), Taif University, Taif, Saudi Arabia. M.Y. thanks to the Indian Council of Medical Research for the award of Senior Research Fellowship (F. No.45/54/2018-PHA/BMS/OL). M.I.H. acknowledges the Council of Scientific and Industrial Research for financial support [Project No. 27(0368)/20/EMR-II]. The authors sincerely thank the Department of Science and Technology, Government of India, for the FIST support (FIST program No. SR/FST/LSII/2020/782).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Boman, H. Antibacterial peptides: Basic facts and emerging concepts. J. Intern. Med. 2003, 254, 197–215. [Google Scholar] [CrossRef] [PubMed]
  2. Szczykutowicz, J.; Tkaczuk-Wlach, J.; Ferens-Sieczkowska, M. Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process. Int. J. Mol. Sci. 2021, 22, 7331. [Google Scholar] [CrossRef] [PubMed]
  3. Roberts, L.M.; Schwarz, B.; Speranza, E.; Leighton, I.; Wehrly, T.; Best, S.; Bosio, C.M. Pulmonary infection induces persistent, pathogen-specific lipidomic changes influencing trained immunity. iScience 2021, 24, 103025. [Google Scholar] [CrossRef] [PubMed]
  4. Grimble, R.F.; Grimble, G.K. Immunonutrition: Role of sulfur amino acids, related amino acids, and polyamines. Nutrition 1998, 14, 605–610. [Google Scholar] [CrossRef]
  5. Dumas, A.; Knaus, U.G. Raising the ‘Good’ Oxidants for Immune Protection. Front. Immunol. 2021, 12, 698042. [Google Scholar] [CrossRef]
  6. Rogan, M.P.; Geraghty, P.; Greene, C.M.; O’Neill, S.J.; Taggart, C.C.; McElvaney, N.G. Antimicrobial proteins and polypeptides in pulmonary innate defence. Respir. Res. 2006, 7, 29. [Google Scholar] [CrossRef] [Green Version]
  7. Schenkels, L.C.; Veerman, E.C.; Nieuw Amerongen, A.V. EP-GP and the lipocalin VEGh, two different human salivary 20-kDa proteins. J. Dent. Res. 1995, 74, 1543–1550. [Google Scholar] [CrossRef]
  8. Sivakumar, S.; Mirels, L.; Miranda, A.J. Hand, AR Secretory protein expression patterns during rat parotid gland development. Anat. Rec. Off. Publ. Am. Assoc. Anat. 1998, 252, 485–497. [Google Scholar] [CrossRef]
  9. Chiu, W.W.C.; Chamley, L.W. Human seminal plasma prolactin-inducible protein is an immunoglobulin G-binding protein. J. Reprod. Immunol. 2003, 60, 97–111. [Google Scholar] [CrossRef]
  10. Gaubin, M.; Autiero, M.; Basmaciogullari, S.; Métivier, D.; Misëhal, Z.; Culerrier, R.; Oudin, A.; Guardiola, J.; Piatier-Tonneau, D. Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells. J. Immunol. 1999, 162, 2631–2638. [Google Scholar]
  11. Witkin, S.S.; Richards, J.M.; Bongiovanni, A.M.; Zelikovsky, G. An IgG-Fc binding protein in seminal fluid. Am. J. Reprod. Immunol. 1983, 3, 23–27. [Google Scholar] [CrossRef] [PubMed]
  12. Bronson, R.A. Antisperm antibodies: A critical evaluation and clinical guidelines. J. Reprod. Immunol. 1999, 45, 159–189. [Google Scholar] [CrossRef]
  13. Rathman, W.; Van, M.Z.; den Keybus Van, P.; Bank, R.; Veerman, E. Nieuw, AA Isolation and characterization of three non-mucinous human salivary proteins with affinity for hydroxyapatite. J. Biol. Buccale 1989, 17, 199–208. [Google Scholar]
  14. Simard, J.; Hatton, A.C.; Labrief, C.; Dauvois, S.; Zhao, H.F.; Haagensen, D.E.; Labrie, F. Inhibitory effect of estrogens on GCDFP-15 mRNA levels and secretion in ZR-75-1 human breast cancer cells. Mol. Endocrinol. 1989, 3, 694–702. [Google Scholar] [CrossRef] [Green Version]
  15. Blanchard, A.A.; Nistor, F.E.; Castaneda, D.; Martin, G.G.; Hicks, F.; Amara, R.P.C.; Shiu, Y.M. Generation and initial characterization of the prolactin-inducible protein (PIP) null mouse: Accompanying global changes in gene expression in the submandibular gland. Can. J. Physiol. Pharmacol. 2009, 87, 859–872. [Google Scholar] [CrossRef] [PubMed]
  16. Hassan, M.I.; Waheed, A.; Yadav, S.; Singh, T.; Ahmad, F. Prolactin inducible protein in cancer, fertility and immunoregulation: Structure, function and its clinical implications. Cell. Mol. Life Sci. 2009, 66, 447–459. [Google Scholar] [CrossRef] [PubMed]
  17. Sharif, R.; Bak-Nielsen, S.; Sejersen, H.; Ding, K.; Hjortdal, J.; Karamichos, D. Prolactin-Induced Protein is a novel biomarker for Keratoconus. Exp. Eye Res. 2019, 179, 55–63. [Google Scholar] [CrossRef] [PubMed]
  18. Mano, F.; Takimoto, H.; Oe, M.; Chang, K.-c.; Mano, T.; Yoshida, Y. Proteomic analysis of dacryoliths from patients with or without topical Rebamipide treatment. Biomed. Hub 2018, 3, 1–11. [Google Scholar] [CrossRef]
  19. Urbaniak, A.; Jablonska, K.; Podhorska-Okolow, M.; Ugorski, M.; Dziegiel, P. Prolactin-induced protein (PIP)-characterization and role in breast cancer progression. Am. J. Cancer Res. 2018, 8, 2150–2164. [Google Scholar]
  20. Haagensen, D.E., Jr.; Mazoujian, G.; Dilley, W.G.; Pedersen, C.E.; Kister, S.J.; Wells Jr, S.A. Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein. J. Natl. Cancer Inst. 1979, 62, 239–247. [Google Scholar]
  21. Murphy, L.C.; Tsuyuki, D.; Myal, Y.; Shiu, R. Isolation and sequencing of a cDNA clone for a prolactin-inducible protein (PIP). Regulation of PIP gene expression in the human breast cancer cell line, T-47D. J. Biol. Chem. 1987, 262, 15236–15241. [Google Scholar] [CrossRef]
  22. Hassan, M.I.; Kumar, V.; Singh, T.P.; Yadav, S. Purification and characterization of zinc α2-glycoprotein-Prolactin inducible protein complex from human seminal plasma. J. Sep. Sci. 2008, 31, 2318–2324. [Google Scholar] [CrossRef] [PubMed]
  23. Hassan, M.I.; Bilgrami, S.; Kumar, V.; Singh, N.; Yadav, S.; Kaur, P.; Singh, T. Crystal structure of the novel complex formed between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma. J. Mol. Biol. 2008, 384, 663–672. [Google Scholar] [CrossRef] [PubMed]
  24. Hassan, I.; Ahmad, F. Structural diversity of class I MHC-like molecules and its implications in binding specificities. Adv. Protein Chem. Struct. Biol. 2011, 83, 223–270. [Google Scholar] [CrossRef]
  25. Léon CPM, S.; Johann, S.; Els, W.-W.; Ingde, S.E.; Enno, V.; Arie, A.V. Identity of human extra parotid glycoprotein (EP-GP) with secretory actin binding protein (SABP) and its biological properties. Biol. Chem. 1994, 375, 609–615. [Google Scholar] [CrossRef] [PubMed]
  26. Schaller, J.; Akiyama, K.; Kimura, H.; Hess, D.; Affolter, M.; Rickli, E.E. Primary structure of a new actin-binding protein from human seminal plasma. Eur. J. Biochem. 1991, 196, 743–750. [Google Scholar] [CrossRef] [PubMed]
  27. Caputo, E.; Carratore, V.; Ciullo, M.; Tiberio, C.; Mani, J.C.; Piatier-Tonneau, D.; Guardiola, J. Biosynthesis and immunobiochemical characterization of gp17/GCDFP-15: A glycoprotein from seminal vesicles and from breast tumors, in HeLa cells and in Pichia pastoris yeast. Eur. J. Biochem. 1999, 265, 664–670. [Google Scholar] [CrossRef] [Green Version]
  28. Terceiro, L.E.L.; Blanchard, A.A.A.; Edechi, C.A.; Fresnoza, A.; Triggs-Raine, B.; Leygue, E.; Myal, Y. Generation of prolactin-inducible protein (Pip) knockout mice by CRISPR/Cas9-mediated gene engineering. Can. J. Physiol. Pharm. 2021, 100, 86–91. [Google Scholar] [CrossRef]
  29. Ihedioha, O.C.; Shiu, R.P.; Uzonna, J.E.; Myal, Y. Prolactin-Inducible Protein: From Breast Cancer Biomarker to Immune Modulator-Novel Insights from Knockout Mice. DNA Cell Biol. 2016, 35, 537–541. [Google Scholar] [CrossRef]
  30. Li, X.; Zhang, Y.; Jia, L.; Xing, Y.; Zhao, B.; Sui, L.; Liu, D.; Xu, X. Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells. Med. Sci. Monit. 2021, 27, e930610. [Google Scholar] [CrossRef]
  31. Kallo, G.; Varga, A.K.; Szabo, J.; Emri, M.; Tozser, J.; Csutak, A.; Csosz, E. Reduced Level of Tear Antimicrobial and Immunomodulatory Proteins as a Possible Reason for Higher Ocular Infections in Diabetic Patients. Pathogens 2021, 10, 883. [Google Scholar] [CrossRef] [PubMed]
  32. Li, J.; Liu, D.; Mou, Z.; Ihedioha, O.C.; Blanchard, A.; Jia, P.; Myal, Y.; Uzonna, J.E. Deficiency of prolactin-inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice. Eur. J. Immunol. 2015, 45, 1082–1091. [Google Scholar] [CrossRef] [PubMed]
  33. Nistor, A.; Bowden, G.; Blanchard, A.; Myal, Y. Influence of mouse prolactin-inducible protein in saliva on the aggregation of oral bacteria. Oral. Microbiol. Immunol. 2009, 24, 510–513. [Google Scholar] [CrossRef] [PubMed]
  34. Gulzar, M.; Ali, S.; Khan, F.I.; Khan, P.; Taneja, P.; Hassan, M.I. Binding mechanism of caffeic acid and simvastatin to the integrin linked kinase for therapeutic implications: A comparative docking and MD simulation studies. J. Biomol. Struct. Dyn. 2019, 37, 4327–4337. [Google Scholar] [CrossRef] [PubMed]
  35. Yousuf, M.; Shamsi, A.; Khan, P.; Shahbaaz, M.; AlAjmi, M.F.; Hussain, A.; Hassan, G.M.; Islam, A.; Rizwanul Haque, Q.M.; Hassan, M. Ellagic acid controls cell proliferation and induces apoptosis in breast cancer cells via inhibition of cyclin-dependent kinase 6. Int. J. Mol. Sci. 2020, 21, 3526. [Google Scholar] [CrossRef] [PubMed]
  36. Khan, P.; Idrees, D.; Moxley, M.A.; Corbett, J.A.; Ahmad, F.; von Figura, G.; Sly, W.S.; Waheed, A.; Hassan, M.I. Luminol-based chemiluminescent signals: Clinical and non-clinical application and future uses. Appl. Biochem. Biotechnol. 2014, 173, 333–355. [Google Scholar] [CrossRef] [Green Version]
  37. Ali, A.; Sultan, I.; Mondal, A.H.; Siddiqui, M.T.; Gogry, F.A.; Haq, Q.M. Lentic and effluent water of Delhi-NCR: A reservoir of multidrug-resistant bacteria harbouring blaCTX-M, blaTEM and blaSHV type ESBL genes. J. Water Health 2021, 19, 592–603. [Google Scholar] [CrossRef]
  38. Wayne, P. National committee for clinical laboratory standards. Perform. Stand. Antimicrob. Disc. Susceptibility Test. 2002, 12, 1–53. [Google Scholar]
  39. Djouossi, M.G.; Ngnokam, D.; Kuiate, J.-R.; Tapondjou, L.A.; Harakat, D.; Voutquenne-Nazabadioko, L. Antimicrobial and antioxidant flavonoids from the leaves of Oncoba spinosa Forssk. (Salicaceae). BMC Complement. Altern. Med. 2015, 15, 134. [Google Scholar] [CrossRef] [Green Version]
  40. Rather, L.J.; Zhou, Q.; Ali, A.; Haque, Q.M.R.; Li, Q. Valorization of agro-industrial waste from peanuts for sustainable natural dye production: Focus on adsorption mechanisms, ultraviolet protection, and antimicrobial properties of dyed wool fabric. ACS Food Sci. Technol. 2021, 1, 427–442. [Google Scholar] [CrossRef]
  41. Vaidya, M.Y.; McBain, A.J.; Butler, J.A.; Banks, C.E.; Whitehead, K.A. Antimicrobial efficacy and synergy of metal ions against Enterococcus faecium, Klebsiella pneumoniae and Acinetobacter baumannii in planktonic and biofilm phenotypes. Sci. Rep. 2017, 7, 5911. [Google Scholar] [CrossRef] [PubMed]
  42. Shabbir, M.; Rather, L.J.; Azam, M.; Haque, Q.M.R.; Khan, M.A.; Mohammad, F. Antibacterial functionalization and simultaneous coloration of wool fiber with the application of plant-based dyes. J. Nat. Fibers 2020, 17, 437–449. [Google Scholar] [CrossRef]
  43. Zhu, Y.; Ramasamy, M.; Yi, D.K. Antibacterial activity of ordered gold nanorod arrays. ACS Appl. Mater. Interfaces 2014, 6, 15078–15085. [Google Scholar] [CrossRef] [PubMed]
  44. Thakur, D.; Ta, Q.T.H.; Noh, J.S. Photon-Induced Superior Antibacterial Activity of Palladium-Decorated, Magnetically Separable Fe3O4/Pd/mpg-C3N4 Nanocomposites. Molecules 2019, 24, 3888. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Akter, S.; Huq, M.A. Biologically rapid synthesis of silver nanoparticles by Sphingobium sp. MAH-11(T) and their antibacterial activity and mechanisms investigation against drug-resistant pathogenic microbes. Artif. Cells Nanomed. Biotechnol. 2020, 48, 672–682. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Tan, M.A.; Castro, S.G.; Oliva, P.M.P.; Yap, P.R.J.; Nakayama, A.; Magpantay, H.D.; Dela Cruz, T.E.E. Biodiscovery of antibacterial constituents from the endolichenic fungi isolated from Parmotrema rampoddense. 3 Biotech 2020, 10, 212. [Google Scholar] [CrossRef] [PubMed]
  47. Chandrasekaran, G.; Lee, Y.C.; Park, H.; Wu, Y.; Shin, H.J. Antibacterial and Antifungal Activities of Lectin Extracted from Fruiting Bodies of the Korean Cauliflower Medicinal Mushroom, Sparassis latifolia (Agaricomycetes). Int. J. Med. Mushrooms 2016, 18, 291–299. [Google Scholar] [CrossRef]
  48. Govindaraju, S.; Samal, M.; Yun, K. Superior antibacterial activity of GlcN-AuNP-GO by ultraviolet irradiation. Mater. Sci. Eng. C Mater. Biol. Appl. 2016, 69, 366–372. [Google Scholar] [CrossRef]
  49. Huq, M.A.; Akter, S. Biosynthesis, Characterization and Antibacterial Application of Novel Silver Nanoparticles against Drug Resistant Pathogenic Klebsiella pneumoniae and Salmonella Enteritidis. Molecules 2021, 26, 5996. [Google Scholar] [CrossRef]
  50. Kloezen, W.; Melchers, R.J.; Georgiou, P.-C.; Mouton, J.W.; Meletiadis, J. Activity of Cefepime in Combination with the novel ß-Lactamase inhibitor taniborbactam (VNRX-5133) against ESBL-producing isolates in in vitro checkerboard assays. Antimicrob. Agents Chemother. 2021, 65, e02338-20. [Google Scholar] [CrossRef]
  51. Wang, T.; Zou, C.; Wen, N.; Liu, X.; Meng, Z.; Feng, S.; Zheng, Z.; Meng, Q.; Wang, C. The effect of structural modification of antimicrobial peptides on their antimicrobial activity, hemolytic activity, and plasma stability. J. Pept. Sci. 2021, 27, e3306. [Google Scholar] [CrossRef] [PubMed]
  52. Fahimmunisha, B.A.; Ishwarya, R.; AlSalhi, M.S.; Devanesan, S.; Govindarajan, M.; Vaseeharan, B. Green fabrication, characterization and antibacterial potential of zinc oxide nanoparticles using Aloe socotrina leaf extract: A novel drug delivery approach. J. Drug Deliv. Sci. Technol. 2020, 55, 101465. [Google Scholar] [CrossRef]
  53. Lee, S.Y.; So, Y.J.; Shin, M.S.; Cho, J.Y.; Lee, J. Antibacterial effects of afzelin isolated from Cornus macrophylla on Pseudomonas aeruginosa, a leading cause of illness in immunocompromised individuals. Molecules 2014, 19, 3173–3180. [Google Scholar] [CrossRef] [PubMed]
  54. Park, S.Y.; Lee, H.U.; Lee, Y.C.; Kim, G.H.; Park, E.C.; Han, S.H.; Lee, J.G.; Choi, S.; Heo, N.S.; Kim, D.L.; et al. Wound healing potential of antibacterial microneedles loaded with green tea extracts. Mater. Sci. Eng. C Mater. Biol. Appl. 2014, 42, 757–762. [Google Scholar] [CrossRef] [PubMed]
  55. Mahajan, P.G.; Dige, N.C.; Suryawanshi, S.B.; Dalavi, D.K.; Kamble, A.A.; Bhopate, D.P.; Kadam, A.N.; Kondalkar, V.V.; Kolekar, G.B.; Patil, S.R. FRET Between Riboflavin and 9-Anthraldehyde Based Fluorescent Organic Nanoparticles Possessing Antibacterial Activity. J. Fluoresc. 2018, 28, 207–215. [Google Scholar] [CrossRef]
  56. Schenkels, L.C.; Walgreen-Weterings, E.; Oomen, L.C.; Bolscher, J.G.; Veerman, E.C.; Nieuw Amerongen, A.V. In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species. Biol. Chem. 1997, 378, 83–88. [Google Scholar] [CrossRef] [PubMed]
  57. Priyadarsini, S.; Hjortdal, J.; Sarker-Nag, A.; Sejersen, H.; Asara, J.M.; Karamichos, D. Gross cystic disease fluid protein-15/prolactin-inducible protein as a biomarker for keratoconus disease. PLoS ONE 2014, 9, e113310. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Konaté, K.; Mavoungou, J.F.; Lepengué, A.N.; Aworet-Samseny, R.R.; Hilou, A.; Souza, A.; Dicko, M.H.; M’Batchi, B. Antibacterial activity against β-lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: Fractional Inhibitory Concentration Index (FICI) determination. Ann. Clin. Microbiol. Antimicrob. 2012, 11, 1–12. [Google Scholar] [CrossRef] [Green Version]
  59. Konaté, K.; Hilou, A.; Mavoungou, J.F.; Lepengué, A.N.; Souza, A.; Barro, N.; Datté, J.Y.; M’batchi, B.; Nacoulma, O.G. Antimicrobial activity of polyphenol-rich fractions from Sida alba L. (Malvaceae) against co-trimoxazol-resistant bacteria strains. Ann. Clin. Microbiol. Antimicrob. 2012, 11, 1–6. [Google Scholar] [CrossRef] [Green Version]
  60. Yu, J.H.; Lim, J.A.; Chang, H.J.; Park, J.H. Characteristics and Lytic Activity of Phage-Derived Peptidoglycan Hydrolase, LysSAP8, as a Potent Alternative Biocontrol Agent for Staphylococcus aureus. J. Microbiol. Biotechnol. 2019, 29, 1916–1924. [Google Scholar] [CrossRef]
  61. Kang, S.M.; Jin, C.; Kim, D.H.; Park, S.J.; Han, S.W.; Lee, B.J. Structure-based design of peptides that trigger Streptococcus pneumoniae cell death. FEBS J. 2021, 288, 1546–1564. [Google Scholar] [CrossRef] [PubMed]
  62. Singh, S.B.; Young, K.; Miesel, L. Screening strategies for discovery of antibacterial natural products. Expert Rev. Anti-Infect. Ther. 2011, 9, 589–613. [Google Scholar] [CrossRef] [PubMed]
  63. Park, J.; Lee, Y.; Hwang, Y.; Cho, S. Interdigitated and Wave-Shaped Electrode-Based Capacitance Sensor for Monitoring Antibiotic Effects. Sensors 2020, 20, 5237. [Google Scholar] [CrossRef] [PubMed]
  64. Harms, A.; Maisonneuve, E.; Gerdes, K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science 2016, 354, aaf4268. [Google Scholar] [CrossRef]
  65. Cooley, L.; Teng, J. Anaerobic resistance: Should we be worried? Curr. Opin. Infect. Dis. 2019, 32, 523–530. [Google Scholar] [CrossRef] [PubMed]
  66. Mun, Y.S.; Hwang, Y.J. Novel spa and Multi-Locus Sequence Types (MLST) of Staphylococcus Aureus Samples Isolated from Clinical Specimens in Korean. Antibiotics 2019, 8, 202. [Google Scholar] [CrossRef] [Green Version]
  67. Kim, H.; Shin, J.Y.; Lee, Y.S.; Yun, S.P.; Maeng, H.J.; Lee, Y. Brain Endothelial P-Glycoprotein Level Is Reduced in Parkinson’s Disease via a Vitamin D Receptor-Dependent Pathway. Int. J. Mol. Sci. 2020, 21, 8538. [Google Scholar] [CrossRef] [PubMed]
  68. Choi, Y.I.; Lee, S.M.; Chung, J.W.; Kim, K.O.; Kwon, K.A.; Kim, Y.J.; Kim, J.H.; Jeong, J.Y.; Park, D.K. Therapeutic Potential of Sitafloxacin as a New Drug Candidate for Helicobacter Eradication in Korea: An In Vitro Culture-Based Study. Antibiotics 2021, 10, 1242. [Google Scholar] [CrossRef] [PubMed]
  69. Dwivedi, G.R.; Rai, R.; Pratap, R.; Singh, K.; Pati, S.; Sahu, S.N.; Kant, R.; Darokar, M.P.; Yadav, D.K. Drug resistance reversal potential of multifunctional thieno[3,2-c]pyran via potentiation of antibiotics in MDR P. aeruginosa. Biomed. Pharm. 2021, 142, 112084. [Google Scholar] [CrossRef]
  70. Tung, T.T.; Tripathi, K.M.; Kim, T.; Krebsz, M.; Pasinszki, T.; Losic, D. Carbon nanomaterial sensors for cancer and disease diagnosis. In Carbon Nanomaterials for Bioimaging, Bioanalysis, and Therapy; Wiley: Hoboken, NJ, USA, 2018; pp. 167–193. [Google Scholar]
  71. Zhong, L.; Liu, H.; Samal, M.; Yun, K. Synthesis of ZnO nanoparticles-decorated spindle-shaped graphene oxide for application in synergistic antibacterial activity. J. Photochem. Photobiol. B 2018, 183, 293–301. [Google Scholar] [CrossRef] [PubMed]
  72. Tripathi, K.M.; Ahn, H.T.; Chung, M.; Le, X.A.; Saini, D.; Bhati, A.; Sonkar, S.K.; Kim, M.I.; Kim, T. N, S, and P-Co-doped Carbon Quantum Dots: Intrinsic Peroxidase Activity in a Wide pH Range and Its Antibacterial Applications. ACS Biomater. Sci. Eng. 2020, 6, 5527–5537. [Google Scholar] [CrossRef] [PubMed]
  73. Singh, S.; Verma, S.; Yadav, D.K.; Kumar, A.; Tyagi, R.; Gupta, P.; Bawankule, D.U.; Darokar, M.P.; Srivastava, S.K.; Kalra, A. The Bioactive Potential of Culturable Fungal Endophytes Isolated from the Leaf of Catharanthus roseus (L.) G. Don. Curr. Top. Med. Chem. 2021, 21, 895–907. [Google Scholar] [CrossRef] [PubMed]
  74. Kovarova-Kovar, K.; Egli, T. Growth kinetics of suspended microbial cells: From single-substrate-controlled growth to mixed-substrate kinetics. Microbiol. Mol. Biol. Rev. 1998, 62, 646–666. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Goo, B.G.; Hwang, Y.J.; Park, J.K. Bacillus thuringiensis: A specific gamma-cyclodextrin producer strain. Carbohydr. Res. 2014, 386, 12–17. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Cloning, expression, and purification of PIP: (A) Amplification of PIP gene through PCR. Lane 1: Marker; Lane 2 and 3 are amplified products of the PIP gene. (B) Lane 1: purified pET28a+ plasmid, Lane 2: pET28a+ plasmid digested with NcoI and XhoI. (C) Restriction digestion studies of PIP expression clone, Lane 1: marker, Lane2: digested pET28a+-PIP construct with NcoI and XhoI, Lane 3: undigested pET28a+-PIP construct. (D) Expression of recombinant PIP using E. coli as an expression host showing un-induced and IPTG-induced bacterial culture having PIP construct. (E) Elution profile of recombinant PIP protein: Lane 1: Marker, Lane 2: supernatant from whole cell lysate before binding to Ni-NTA column; Lane 3: supernatant after Ni-NTA binding; Lane 4: elution from 20 mM imidazole elution buffer; Lane 5: elution from 50 mM imidazole buffer; Lane 6: elution from 200 mM imidazole buffer; Lane 7: elution from 300 mM imidazole buffer; Lane 8: elution from 500 mM imidazole buffer. (F) PIP elution profile from ion-exchange chromatography. (G) The 15% SDS-PAGE for PIP elutions; Lane 1: Marker, Lane 2: impure PIP before loading to ion-exchange chromatography, Lane 3–5: showing increasing concentration gradients (5 µg, 10 µg and 15 µg, respectively) of purified PIP eluted from ion-exchange chromatography.
Figure 1. Cloning, expression, and purification of PIP: (A) Amplification of PIP gene through PCR. Lane 1: Marker; Lane 2 and 3 are amplified products of the PIP gene. (B) Lane 1: purified pET28a+ plasmid, Lane 2: pET28a+ plasmid digested with NcoI and XhoI. (C) Restriction digestion studies of PIP expression clone, Lane 1: marker, Lane2: digested pET28a+-PIP construct with NcoI and XhoI, Lane 3: undigested pET28a+-PIP construct. (D) Expression of recombinant PIP using E. coli as an expression host showing un-induced and IPTG-induced bacterial culture having PIP construct. (E) Elution profile of recombinant PIP protein: Lane 1: Marker, Lane 2: supernatant from whole cell lysate before binding to Ni-NTA column; Lane 3: supernatant after Ni-NTA binding; Lane 4: elution from 20 mM imidazole elution buffer; Lane 5: elution from 50 mM imidazole buffer; Lane 6: elution from 200 mM imidazole buffer; Lane 7: elution from 300 mM imidazole buffer; Lane 8: elution from 500 mM imidazole buffer. (F) PIP elution profile from ion-exchange chromatography. (G) The 15% SDS-PAGE for PIP elutions; Lane 1: Marker, Lane 2: impure PIP before loading to ion-exchange chromatography, Lane 3–5: showing increasing concentration gradients (5 µg, 10 µg and 15 µg, respectively) of purified PIP eluted from ion-exchange chromatography.
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Figure 2. Zone of inhibition assay. (A) Representative images of culture plates showing PIP mediated growth inhibition of Bacillus subtilis, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa. (B) Quantification of ZOI studies for Bacillus subtilis, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa.
Figure 2. Zone of inhibition assay. (A) Representative images of culture plates showing PIP mediated growth inhibition of Bacillus subtilis, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa. (B) Quantification of ZOI studies for Bacillus subtilis, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa.
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Figure 3. Zone of inhibition assay: (A) Representative images of culture plates showing PIP mediated growth inhibition of Citrobacter sp., Bacillus sp., E. Coli (SD6), and C. werkmanii; (B) Quantification of ZOI studies for Citrobacter sp., Bacillus sp., E. coli (SD6), and C. werkmanii.
Figure 3. Zone of inhibition assay: (A) Representative images of culture plates showing PIP mediated growth inhibition of Citrobacter sp., Bacillus sp., E. Coli (SD6), and C. werkmanii; (B) Quantification of ZOI studies for Citrobacter sp., Bacillus sp., E. coli (SD6), and C. werkmanii.
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Figure 4. Growth kinetics study of (A) E. coli, (B) P. aeruginosa at different MIC concentrations of PIP. Ampicillin (AMP) was taken as a positive control.
Figure 4. Growth kinetics study of (A) E. coli, (B) P. aeruginosa at different MIC concentrations of PIP. Ampicillin (AMP) was taken as a positive control.
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Table
Table 1. MIC and MBC Values (µg/mL) of PIP and AMP against bacterial strains.
Table 1. MIC and MBC Values (µg/mL) of PIP and AMP against bacterial strains.
IsolatesMIC MBC MBC/MIC
PIP
(µg/mL)		Standard Strains 443E. coli321284
736B. subtilis642564
902S. aureus1285124
2453P. aeruginosa641282
Environmental
MDR strains 		HK 106Citrobacter sp.128>10248
LG 1Bacillus sp.645128
SD 6E.coli51210242
SH 52B. werkmanii1285124
Amp
(µg/mL)		Standard Strains 443E. coli1--
736B. subtilis0.5--
902S. aureus0.25--
2453P. aeruginosa4--
MDR strains HK 106Citrobacter sp.128--
LG 1Bacillus sp.256--
SD 6E.coli1024--
SH 52B. werkmanii1024--
Minimal bactericidal concentration values for the PIP against eight studied bacterial isolates. Citrobacter sp. (HK106/MT576965) shows maximum MBC value (>1024 µg/mL), while Pseudomonas aeruginosa (2453) and E. coli (443) shows minimal MBC value (128 µg/mL) among the tested isolates.
Table
Table 2. Zone of inhibition (in mm) as measured around the disk of various concentrations of compound.
Table 2. Zone of inhibition (in mm) as measured around the disk of various concentrations of compound.
Types of Bacterial IsolatesIsolates CodeBacterial StrainZone of Inhibition at Different Concentrations of Test Compound
MIC/2MIC2MIC
Standard
Bacterial Strains		443E. coli162026
736B. subtilis8912
902S. aureus81317
2453P. aeruginosa6813
Environmental
MDR Bacterial strains		HK 106Citrtobacter sp.6810
LG 1Bacillus sp.81214
SD 6E. coli81014
SH 52C. werkmanii7912
Table
Table 3. Synergistic antibacterial activity of PIP with AMP.
Table 3. Synergistic antibacterial activity of PIP with AMP.
Bacterial StrainMIC Alone (μg/mL)MIC in Combination (μg/mL)FICI *Mode of Interaction
PIPAMPPIPAMP
E.coli (443)32120.1250.187Synergistic
P. aeruginosa (2453)1284810.312Synergistic
E.coli (SD 6)51210245125121.5Indifferent
Citrobacter werkmanii
(SH 52)		1281024120.0097Synergistic
A FICI of ≤ 0.5 was defined as synergy, a FICI of > 0.5 but ≤ 4.0 was defined as no interaction, and a FICI of > 4.0 was defined as antagonism. * FICI: fractional inhibitory concentration index.
Table
Table 4. Resistance pattern of environmental isolates.
Table 4. Resistance pattern of environmental isolates.
IsolatesStrainAccession NoResistance PatternMIC (µg/mL) for AMPSite
HK 106Citrtobacter sp.MT576965AMP, ETP, IPM, CX, P/T CZ, PB, AK, CIP, CL, TR128Hauz Khas lake
LG 1Bacillus sp.MT576690AMP, CX, A/S, RIF, CZ, TR256Lodhi Garden Pond
SD 6Escherichia coliMT577556AMP, IMP, CX, RIF, CZ, PB, AK, TE, CIP, LE1024Shaheen Bagh Drain
SH 52Citrtobacter werkmaniiMN267555ETP, CX, RIF, CZ, PB, CL, TR1024Ghazipur Slaughterhouse effluent
AMP: Ampicillin, A/S: Ampicillin/Sulbactam, CX: Cefoxitin, IPM: Imipenem, P/T: Piperacillin/Tazobactam, CZ: Cefazolin, ETP: Ertapenem, RIF: Rifampicin AK: Amikacin, CL: Colistin, PB: Polymyxin B, TE: Tetracycline, CIP: Ciprofloxacin, LE: Levofloxacin, TR: Trimethoprim.
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Yousuf, M.; Ali, A.; Khan, P.; Anjum, F.; Elasbali, A.M.; Islam, A.; Yadav, D.K.; Shafie, A.; Rizwanul Haque, Q.M.; Hassan, M.I. Insights into the Antibacterial Activity of Prolactin-Inducible Protein against the Standard and Environmental MDR Bacterial Strains. Microorganisms 2022, 10, 597. https://doi.org/10.3390/microorganisms10030597

AMA Style

Yousuf M, Ali A, Khan P, Anjum F, Elasbali AM, Islam A, Yadav DK, Shafie A, Rizwanul Haque QM, Hassan MI. Insights into the Antibacterial Activity of Prolactin-Inducible Protein against the Standard and Environmental MDR Bacterial Strains. Microorganisms. 2022; 10(3):597. https://doi.org/10.3390/microorganisms10030597

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Yousuf, Mohd, Asghar Ali, Parvez Khan, Farah Anjum, Abdelbaset Mohamed Elasbali, Asimul Islam, Dharmendra Kumar Yadav, Alaa Shafie, Qazi Mohd. Rizwanul Haque, and Md. Imtaiyaz Hassan. 2022. "Insights into the Antibacterial Activity of Prolactin-Inducible Protein against the Standard and Environmental MDR Bacterial Strains" Microorganisms 10, no. 3: 597. https://doi.org/10.3390/microorganisms10030597

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