The Antibacterial Activity of Human Amniotic Membrane against Multidrug-Resistant Bacteria Associated with Urinary Tract Infections: New Insights from Normal and Cancerous Urothelial Models

Abstract

Urinary tract infections (UTIs) represent a serious global health issue, especially due to emerging multidrug-resistant UTI-causing bacteria. Recently, we showed that the human amniotic membrane (hAM) could be a candidate for treatments and prevention of UPEC and Staphylococcus aureus infections. However, its role against multidrug-resistant bacteria, namely methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae, vancomycin-resistant Enterococci (VRE), carbapenem-resistant Acinetobacter baumannii, and Pseudomonas aeruginosa has not yet been thoroughly explored. Here, we demonstrate for the first time that the hAM homogenate had antibacterial activity against 7 out of 11 tested multidrug-resistant strains, the greatest effect was on MRSA. Using novel approaches, its activity against MRSA was further evaluated in a complex microenvironment of normal and cancerous urinary bladder urothelia. Even short-term incubation in hAM homogenate significantly decreased the number of bacteria in MRSA-infected urothelial models, while it did not affect the viability, number, and ultrastructure of urothelial cells. The hAM patches had no antibacterial activity against any of the tested strains, which further exposes the importance of the hAM preparation. Our study substantially contributes to basic knowledge on the antibacterial activity of hAM and reveals its potential to be used as an antibacterial agent against multidrug-resistant bacteria.

Keywords: MRSA; amniotic membrane homogenate; antibiotic resistance; antimicrobial activity; electron microscopy; multidrug-resistant bacteria; urinary bladder; urothelial cells.

Figure 1

The human amniotic membrane (hAM) patches have no antibacterial activity against tested multidrug-resistant bacteria. Neither the application of fresh-hAM (f-hAM) patches (A–D, I–K, O–R) nor the application of cryopreserved hAM (c-hAM) patches (E–H,L–N,S–V) resulted in an inhibition zone in any of the tests. Data were obtained from three independent replications of experiments using three biological samples of hAM and three technical repeats for each biological sample and each independent replication of the experiment. Scale bars: 10 mm.

Figure 2

The f-hAM and c-hAM homogenates have antibacterial activity against seven out of 11 tested strains. (A,B,E,F,I,L) The application of f-hAM and c-hAM homogenates resulted in an inhibition zone in all the tests. (C,D,G,H) The application of f-hAM and c-hAM homogenates resulted in an inhibition zone in 75% of all the performed tests. (J,K,M,N) The application of f-hAM and c-hAM homogenates resulted in an inhibition zone in 25% of the performed tests. (O–V) The application of f-hAM and c-hAM homogenates did not result in an inhibition zone in any of the performed tests. Data were obtained from at least three independent replications of experiments using at least three biological samples of hAM and six technical repeats for each biological sample and each independent replication of the experiment. Scale bars: 10 mm.

Figure 3

The range of antibacterial activity of f-hAM and c-hAM homogenates varies between the multidrug-resistant strains. The average antibacterial activity of f-hAM and c-hAM homogenates against all the susceptible strains is shown. The bars (red) show the mean diameter of the inhibition zone ± SEM (mm) of all the susceptible strains. On average, the application of f-hAM homogenate resulted in a larger inhibition zone than the application of c-hAM homogenate. Furthermore, a larger volume of hAM homogenates applied (10 µl) resulted in a larger inhibition zone than the smaller volume of hAM homogenates (5 µl). (*, **) Antibacterial activity of f-hAM and c-hAM homogenates was detected in 75% (*) or 25% (**) of all the performed tests and only these measurements were included in the mean diameter ± SEM (mm) of the inhibition zones.

Figure 4

Comparison of the antibacterial activity of hAM homogenates and selected antibiotics against the reference strain of methicillin-resistant S. aureus (MRSA). (A,D) The reference strain of MRSA is resistant to trimethoprim/sulfamethoxazole, clindamycin, erythromycin, penicillin, and cefoxitin and is susceptible to linezolid. (B–D) The application of 5 and 10 μL f-hAM and c-hAM homogenates results in an inhibition zone. Scale bars: 10 mm.

Figure 5

The effect of c-hAM homogenate on MRSA-infected biomimetic in vitro models of the normal and cancerous urothelium. (A–H) Scheme of the experiment. The normal porcine urothelial (NPU) and T24 cells were incubated for 3 h in the (A,B) culture medium (control), (C,D) c-hAM homogenate, (E,F) culture medium inoculated with MRSA, (G,H) c-hAM homogenate inoculated with MRSA. (I) The number of bacteria in MRSA-infected samples, incubated in the presence or absence of c-hAM homogenate. The c-hAM homogenate significantly decreased the number of bacteria in biomimetic in vitro models of the normal and cancerous urothelium. * p < 0.05; ** p < 0.001.

Figure 6

The effect of short-term incubation in the c-hAM homogenate and/or MRSA on the viability of biomimetic in vitro models of the normal and cancerous urothelium. The NPU (A) and T24 cells (B) maintained a high cell viability in all conditions and there were no statistically significant differences (p > 0.05) in cell viability between the treated and non-treated NPU and T24 cells. There were also no statistically significant differences in the number of viable cells between the treated and non-treated NPU and T24 cells. Data presented here show the percentage of viable cells ± SEM and the mean number of cells ± SEM for each sample. Data were obtained from three independent replications of experiments using three biological samples of hAM; each experiment was performed in two technical repeats for each condition.

Figure 7

The effect of short-term incubation in the c-hAM homogenate and/or MRSA on the ultrastructure of biomimetic in vitro models of the normal and cancerous urothelium. (A,B,E,F) Short-term (3 h) treatment of the NPU cells with the c-hAM homogenate does not affect the ultrastructure of NPU cells. (C,D,G,H) During the 3 h incubation in the culture medium inoculated with MRSA, individual bacteria or small aggregates of MRSA attached to the surface of NPU cells. During the 3 h incubation in the c-hAM homogenate inoculated with MRSA, a smaller number of individual bacteria attached to the surface of NPU cells. (I,J,M,N) A short-term treatment of the T24 cells with the c-hAM homogenate does not affect the ultrastructure of T24 cells. (K,O) During the 3 h incubation in the culture medium inoculated with MRSA, aggregates of MRSA attached to the surface of the T24 cells and some of them were endocytosed by the T24 cells. (L,P) During the 3 h incubation in the c-hAM homogenate inoculated with MRSA, a smaller number of individual bacteria or small aggregates of bacteria attached to the surface of T24 cells. Large insets framed with white lines (A–D,I–L) show the enlarged areas of the corresponding small white-framed insets. Data were obtained from three independent replications of experiments using three biological samples of hAM. Arrow: An aggregate of MRSA in the endosomal compartment. Scale bars: (A–D,I–L) 10 µm; (enlarged areas in A–D,I–L) 1 µm; (E–H,M–P) 600 nm.