Poe Plague Of Rats
Biofilms empowerment theory social work communities of microorganisms attached to a surface The Controversy Between Abortion And Pro-Life involved by a self-produced Nikes Rhetorical Analysis Essay matrix [ 1718 ]. In nonstandard burial practices, such as mass burialthe body may be positioned arbitrarily. Rats were considered to have biofilms when they were positive in the co-localization technique AB and IHC anti- Leptospira. Empowerment theory social work genera of rodents. A Master Seeks Help. The Controversy Between Abortion And Pro-Life Devil will drop The Controversy Between Abortion And Pro-Life Perpetuum upon slaying. Retrieved October What Are The Three Branches Of Government Gilded Torment Scarab. Hidden Reinforcements.
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Experimentally infected R. Histopathological analysis of the kidneys of naturally infected R. However, it was not possible to detect if those alterations were related to Leptospira infection, once numerous environmental exposures could cause similar alterations [ 9 , 12 ]. During infection in chronic animal models, leptospires form biofilm-like structures during renal tubular colonization [ 13 ], what may explain the antibiotic tolerance observed when the treatment occurs during the disease chronic phase [ 14 ].
Bacteria of the genus Leptospira form biofilms in vitro [ 15 ] and in rural environment [ 16 ]. Biofilms are communities of microorganisms attached to a surface and involved by a self-produced exopolymeric matrix [ 17 , 18 ]. The transcriptome of saprophytic Leptospira biflexa revealed important insights into Leptospira biology during biofilm formation. Of note, there was a great shift in gene regulation during biofilm, as the diminishment of alginate gene expression, upregulation of a putative gene for a virulence-associated protein, and upregulation of outer membrane proteins OMPs encoding genes [ 19 ].
A recent study analyzed in vitro several aspects of the biofilm formed by pathogenic Leptospira and showed that inside the biofilm leptospires are protected against stressful environmental conditions as pH and temperature [ 20 ]. The biofilm lifestyle is ubiquitous in Bacteria. This ubiquity is related to the protection offered by biofilms against harsh environmental and host conditions [ 18 , 21 ]. Biofilms are described as colonization and virulence factors [ 22 ], participating in the pathogenesis of several diseases caused by pathogenic or opportunistic bacteria [ 23 , 24 ]. Bacterial biofilms are associated with many medical chronic conditions as cystic fibrosis pneumonia, dental plaque, and catheter contamination, among others [ 24 — 27 ].
The spirochete Borrelia form biofilms in vitro and in vivo in the skin of infected patients diagnosed with borrelial lymphocytoma [ 28 , 29 ]. Biofilms were also described as crucial for the transmission of Yersinia pestis and host-pathogen interactions in plague, due to biofilm formation in the midgut of fleas [ 30 ]. Knowledge about pathogenic Leptospira biology, host-pathogen interactions, and survival inside the hosts, as well as their transmission mechanisms to other hosts are of outermost importance to understand and counteract the infection in susceptible hosts [ 31 , 32 ].
In this study we demonstrated Leptospira biofilms in the kidneys of naturally infected Rattus norvegicus captured in an urban slum endemic for leptospirosis. We identified the factors associated with renal biofilm formation and characterized the histopathological kidney alterations in natural reservoir rats. Pau da Lima is an urban community with 0. We screened the kidney sections for L. We then applied a questionnaire for histopathological analysis.
For biofilm detection, we used AB pH 2. Then, we washed the samples under tap water for 10 min; added mucicarmine solution for 1 h; washed quickly in non-pyrogenic distilled water, added metanil yellow for 1 min, and washed again in non-pyrogenic distilled water. For AB staining, we deparaffinized the samples in xylol baths twice for 10 min each; immersed in absolute alcohol baths twice for 30 s each and hydrated under tap water. We immersed the slides into Harris hematoxylin for 1 min, rinsed in tap water for 5 min, dehydrated in absolute alcohol baths three times for 3 min each, clarified in two xylol baths, and assembled the slides.
We analyzed biofilm formation only in the kidneys of positive rats for IHC anti- L. As negative controls, we used kidneys of negative rats for L. Once we identified renal tubules positive to IHC anti- L. We analyzed different patterns of leptospiral colonization in R. We inferred colonization intensity by counting the number of IHC positive colonized tubules CTs in a half-kidney cortex. We analyzed the distribution of CTs by performing a qualitative evaluation, registering if CTs were observed isolated in the cortex one CT or agglomerated two or more CTs , and with focal or multifocal distribution.
A non-colonized rat kidney was processed as negative control. We then transferred the samples to new tubes containing the same fixative with or without 0. For this analysis, we used a subsampling of four positive rats for Leptospira infection with renal biofilm and one negative rat as a control. Rats were considered to have biofilms when they were positive in the co-localization technique AB and IHC anti- Leptospira. Sixty-seven rats were analyzed for patterns of leptospiral colonization and 64 rats with positive IHC anti- L.
We used generalized linear model GLM to analyze the risk factors associated with Leptospira infection occurrence and renal biofilm formation in the chronically infected host, R. GLM was also used to analyze demographic data associated with histological characteristics of infected rats. We used R Version 1. All results are available in S1 Table. We captured 87 R. From the 78 rats, 65 were positive for immunohistochemistry IHC anti- Leptospira and were analyzed for the presence of renal biofilm. A Alcian Blue AB positively stained renal tubules red arrows observed in light turquoise blue, indicating the presence of biofilm matrix; insert B with detail of biofilm staining.
Note that the serial sections showed in A and D are from the same region of one rat kidney, evidencing the co-localization of tubules concomitantly positive in AB red arrows and IHC black arrows. Magnification, x However, generalized linear model GLM analysis did not show statistical association between renal biofilm formation and demographic characteristics and leptospiral urine shedding. We analyzed the association of renal biofilm formation with renal colonization patterns. GLM showed that the intensity of colonized tubules increased the chance of renal biofilm formation Table 1. Furthermore, the partial to complete pattern of tubule colonization increased in more than three times the chance of having renal biofilm Table 1. We observed dense leptospiral colonization, forming aggregates inside the renal tubules Fig 2A and 2B —white arrows , with heavy deposition of amorphous extracellular matrix between leptospires, often covering and embedding spirochete bacteria, compatible with biofilm morphology Fig 2A and 2B —red arrows.
When we analyzed the kidneys of infected rats without RR staining, we observed the presence of isolated and agglomerated leptospires inside the renal tubules, without amorphous extracellular matrix in-between and embedded-in Fig 2C and 2D —white arrows. Finally, we did not observe biofilm nor Leptospira in negative controls Fig 2E and 2F. A and B—SEM of colonized kidney with ruthenium red RR showed Leptospira a gglomerates white arrows surrounded by an anionic exopolysaccharidic matrix red arrows inside the renal tubules. Histopathological analysis revealed minimal alterations in the kidneys of rats with renal biofilm Table 2. Immunohistochemical representative images of kidney with A high intensity of colonized tubules CTs ; B low intensity of CTs; C partial to complete pattern of CT lumen occlusion; D marking restricted to the membrane of renal epithelium partial occlusion ; E agglomerated CTs distributed in the renal cortex; F isolated CTs distributed in the cortex.
A, B, C and D: magnification, x E and F: magnification, x The intensity of colonized tubules was associated with demographic data and leptospiral shedding in urine. Male and juvenile rats had a higher chance of having more CT count, while rats captured at valley 4 were more likely to have fewer Table 3. We identified and characterized leptospiral biofilm formation inside the renal tubules of R. By analyzing demographic data and histological patterns of renal colonization by leptospires, we identified the risk factors associated with biofilm formation. Finally, we characterized R.
Reservoir hosts infected with Leptospira and experimentally infected rats chronic model of disease present dense renal tubules colonization [ 9 , 13 ]. Biofilm formation by pathogenic bacteria has been described in other hosts. Borrelia burgdorferi , the causative agent of Lyme disease, are spirochetes capable of forming biofilms in vitro , in the midguts of infected ticks, and in the skin tissue of borrelial lymphocytoma patients [ 28 , 29 , 40 ]. Yersinia pestis , the causative agent of bubonic plague, form biofilm inside the flea gut [ 41 ]. In both cases, biofilm formation is described as having a role in the transmission of the pathogen to the accidental host. Additionally, bacterial biofilms are considered colonization factors since they may contribute to bacterial evasion from the immune system [ 42 — 44 ].
We noticed a positive correlation between the intensity of CT count and biofilm formation, that is, the greater the number of colonized tubules, greater was the chance of renal biofilm formation by pathogenic Leptospira. If we consider the number of CT as a measure of Leptospira population inside the organ, this finding may indicate that biofilm formation in vivo by Leptospira is dependent on leptospiral numbers during renal colonization, suggesting the presence of quorum sensing mechanisms [ 45 ].
Moreover, our data showed that Norway rats with renal biofilm excreted ten times less leptospires in the urine than rats positive for renal infection but negative for biofilms, what we believe is a consequence of leptospires maintenance in the renal biofilm. The majority of animals presenting biofilms had a marking pattern of IHC anti- L. This complete pattern of renal colonization increased in more than three times the chance of renal biofilm development. Furthermore, 21 Norway rats naturally infected and with renal biofilm had agglomerated pattern of CT. Thus, it is possible to hypothesize that single leptospires infect the kidneys diffusely, coming from the circulatory system, migrate through tissue, multiply and colonize into the proximal renal tubules, where they form cell aggregates and biofilms [ 13 ].
The authors observed biofilms formed by a network of leptospires embedded in the extracellular matrix, although they did not use extracellular matrix specific stains. Thibeaux and collaborators analysed leptospiral charge in urine and kidneys of experimentally infected rodents, by qPCR, but did not explore structural aspects of biofilms formed in vivo [ 20 ]. Finally, Yamaguchi and collaborators used TEM to analyze the renal tubules of infected mice and suggested the presence of biofilm-like structures during tubular colonization by pathogenic leptospires; although they did not use specific dyes for biofilms [ 13 ].
We observed that R. SEM using RR revealed the ultrastructure of the tubular biofilms with agglomerates of Leptospira involved in an acidic exopolysaccharidic matrix. Alcian blue, Mayer mucicarmine and ruthenium red are staining methods used to visualize the exopolymeric matrix. AB pH 2. Those stains are commonly used to characterize extracellular matrix of bacterial biofilms, both in in vitro studies [ 28 ] and in in vivo studies [ 27 , 29 , 48 — 50 ].
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