Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine.

Laboratory investigations were conducted to study potential mechanisms for prolonged survival of Pseudomonas aeruginosa in poloxamer-iodine (PxI). P. aeruginosa organisms isolated from PxI and adapted for growth in distilled water or found as part of a mixed microbial population from water in a manufacturing plant did not survive more than 15 s after challenge in stock PxI solution. Batches of PxI were compounded in the laboratory to determine the survival and growth of P. aeruginosa during the various stages of preparation. No P. aeruginosa organisms were recovered from the finished product at 1 min after the addition of iodine-iodide. However, we found P. aeruginosa in PxI 48 h after adding sterile PxI to the inside of a naturally contaminated polyvinyl chloride water distribution pipe. These organisms (10(4) CFU/ml) survived for as long as 98 days in contaminated stock PxI after it was removed from the polyvinyl chloride pipe. Both decreasing the free iodine level through addition of potassium iodide and increasing the free iodine level through dilution of the product resulted in an increased length of survival of P. aeruginosa in contaminated PxI solution. Comparative survival studies with pipes of different composition revealed that other materials may exert an effect similar to polyvinyl chloride. We concluded that polyvinyl chloride and perhaps other materials may play an important role in the survival of P. aeruginosa in iodophors and may be one source of resistant microbial populations when used in manufacturing plants which produce these antimicrobial solutions.     

Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine

Laboratory investigations were conducted to study potential mechanisms for prolonged survival of Pseudomonas aeruginosa in poloxamer-iodine (PxI). P. aeruginosa organisms isolated from PxI and adapted for growth in distilled water or found as part of a mixed microbial population from water in a manufacturing plant did not survive more than 15 s after challenge in stock PxI solution. Batches of PxI were compounded in the laboratory to determine the survival and growth of P. aeruginosa during the various stages of preparation. No P. aeruginosa organisms were recovered from the finished product at 1 min after the addition of iodine-iodide. However, we found P. aeruginosa in PxI 48 h after adding sterile PxI to the inside of a naturally contaminated polyvinyl chloride water distribution pipe. These organisms (10(4) CFU/ml) survived for as long as 98 days in contaminated stock PxI after it was removed from the polyvinyl chloride pipe. Both decreasing the free iodine level through addition of potassium iodide and increasing the free iodine level through dilution of the product resulted in an increased length of survival of P. aeruginosa in contaminated PxI solution. Comparative survival studies with pipes of different composition revealed that other materials may exert an effect similar to polyvinyl chloride. We concluded that polyvinyl chloride and perhaps other materials may play an important role in the survival of P. aeruginosa in iodophors and may be one source of resistant microbial populations when used in manufacturing plants which produce these antimicrobial solutions.     

Biofilm formation in an experimental water distribution system: the contamination of non-touch sensor taps and the implication for healthcare

Hospital tap water is a recognised source of Pseudomonas aeruginosa. UK guidance documents recommend measures to control/minimise the risk of P. aeruginosa in augmented care units but these are based on limited scientific evidence. An experimental water distribution system was designed to investigate colonisation of hospital tap components. P. aeruginosa was injected into 27 individual tap ‘assemblies’. Taps were subsequently flushed twice daily and contamination levels monitored over two years. Tap assemblies were systematically dismantled and assessed microbiologically and the effect of removing potentially contaminated components was determined. P. aeruginosa was repeatedly recovered from the tap water at levels above the augmented care alert level. The organism was recovered from all dismantled solenoid valves with colonisation of the ethylene propylene diene monomer (EPDM) diaphragm confirmed by microscopy. Removing the solenoid valves reduced P. aeruginosa counts in the water to below detectable levels. This effect was immediate and sustained, implicating the solenoid diaphragm as the primary contamination source.     

Evaluation of a Most-Probable-Number Technique for the Enumeration of Pseudomonas aeruginosa

A most-probable-number (MPN) technique was evaluated for detecting and enumerating Pseudomonas aeruginosa in water and wastewater. Both the presumptive and confirmatory media, as described in the 13th edition of Standard Methods for the Examination of Water and Wastewater, as well as modifications of these media were included in evaluations. Various samples of water were tested, namely chlorinated tap water, creek water, and influent to a wastewater treatment plant. Modified media repeatedly gave higher estimated MPNs of P. aeruginosa than media listed in Standard Methods. P. aeruginosa was detected and recovered from all creek water and wastewater samples, but not from tap water samples tested. This organism was determined to be present in as large numbers as the fecal coliforms and in even greater quantities than the fecal streptococci in all samples, whenever MPN estimations were determined from those positive tubes containing the modified confirmatory medium.     

Pseudomonas aeruginosa and Achromobacter sp. Clonal Selection Leads to Successive Waves of Contamination of Water in Dental Care Units

Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs.     

Membrane Filter Technique for Enumeration of Pseudomonas aeruginosa

A membrane filter procedure for the quantitation of Pseudomonas aeruginosa (mPA procedure) has been developed. Through the use of inhibitors and an elevated incubation temperature, the level of background microbial flora was decreased approximately 10,000-fold. Using P. aeruginosa cells suspended in sea water and held for 24 hr, between 70 and 100% of the „viable” cells could be recovered by the mPA procedure. Assay variability was found to be insignificant. The recoveries of P. aeruginosa from surface (fresh and salt) waters, potable waters, and sewage by the mPA procedure exceeded those obtainable by current methods. Subsequent to its development and evaluation, the mPA procedure was used at three other laboratories for the enumeration of P. aeruginosa in potable and recreational waters and in sewage samples. It was found amenable to routine use, and confirmation of typical colonies approached 100%.     

Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria

High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10³ cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10² cfu/100 ml for P. aeruginosa and 2.66 × 10² cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.     

Effect of long-term starvation in salty microcosm on biofilm formation and motility in Pseudomonas aeruginosa

The development of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though P. aeruginosa considered a model organism for the study of biofilm formation. In this investigation, P. aeruginosa ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered P. aeruginosa, differential display “DDRT-PCR” technique was used. The genes speD and ccoN2, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for speD gene suggested that spermidine has a potential role in the adaptive response in P. aeruginosa incubated in long-term stress conditions.     

Ecological responses and remediation ability of water fern (Azolla japonica) to water pollution

The ability of the water fern Azolla japonica to remediate phosphorus (P), nitrogen (N), and iron (Fe) contamination, and its physiological responses to three common sources of water pollution (landfill leachate, swine lagoon sewage, and fish farm sewage) and standard solution were investigated. The biomass, water content, and chlorophyll content of Azolla japonica in each solution were measured, and the concentrations and accumulation rates of polluting elements in the solutions were determined. A. japonica showed over eight-fold increase in biomass within only 20 d in every solution except in swine lagoon sewage, extremely high in N concentration. Consistent chlorophyll and water contents of the plant in most solutions showed that A. japonica can adapt to highly concentrated solutions. N, P, and Fe concentrations of the solutions decreased significantly within the 20 d. In most treatments, A. japonica showed high N accumulation and also showed total uptake of P and Fe from the solutions. In reference to this result, using this species as a phytoremediator plant would have additional benefits of helping maintaining endangered populations of A. japonica. Therefore, the plant??s fast growth, good element remediation efficiency, and conservation needs makes A. japonica a suitable plant species for pollution remediation.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.     

The effects of human tear electrolytes on the viability and hydrogel colonization of Pseudomonas aeruginosa and Staphylococcus aureus

This study was designed to evaluate the effects of the inorganic electrolytes present in human tear film on the viability and colonization of bacteria to hydrogels. Pseudomonas aeruginosa and Staphylococcus aureus were used in these experiments. A D-value test was performed to investigate any bacteriostatic effect by measuring the reduction of viable test microorganisms over time when exposed to the inorganic electrolyte solution. No D-value was calculable for S. aureus in electrolyte solution whereas a D-value of 8.1 h was obtained for P. aeruginosa in electrolyte solution. The D-value data indicate that staphylococci have a greater survivability potential in a hypertonic environment than do pseudomonads. Bacterial adhesion to high water, ionic hydrogels was studied using the Modified Robbins Device (MRD). The data for P. aeruginosa recovered from the lenses showed an 82% decrease in bacterial counts in electrolyte solution as compared to bacteria incubated in control solution. In contrast there were slight increases in S. aureus counts recovered from the lenses. Journal of Industrial Microbiology & Biotechnology (2000) 25, 17–19. Received 12 August 1999/ Accepted in revised form 05 January 2000     

Improved PCR for identification of Pseudomonas aeruginosa

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41?×?10³?copies/μl of cloned amplified target DNA using purified DNA, or 2.7?×?10² colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.     

Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10⁶ CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.     

Bacteriological Evaluation of an Ultra-Pure Water-Distilling System

A prototype distillation and storage system with recycle for producing ultrapure water was monitored for bacteriological contamination during a period of 24 months. Naturally occurring Pseudomonas aeruginosa and P. cepacia were found to grow rapidly to levels of about 10⁵/ml in water taken from the storage reservoir and also in commercially prepared distilled water. The system was found to eliminate bacterial contaminants introduced into the still with the feed water, but the reservoir, once contaminated, remained contaminated during prolonged recycle. After a single treatment with free chlorine, the entire system remained uncontaminated until accidental or purposeful shutdown.     

Alleviation of fungicide-induced phytotoxicity in greengram [Vigna radiata (L.) Wilczek] using fungicide-tolerant and plant growth promoting Pseudomonas strain

This study was designed to explore beneficial plant-associated rhizobacteria exhibiting substantial tolerance against fungicide tebuconazole vis-à-vis synthesizing plant growth regulators under fungicide stressed soils and to evaluate further these multifaceted rhizobacteria for protection and growth promotion of greengram [Vigna radiata (L.) Wilczek] plants against phytotoxicity of tebuconazole. Tebuconazole-tolerant and plant growth promoting bacterial strain PS1 was isolated from mustard (Brassica compestris) rhizosphere and identified as Pseudomonas aeruginosa following 16S rRNA gene sequencing. The P. aeruginosa strain PS1 solubilized phosphate significantly and produced indole acetic acid, siderophores, exo-polysaccharides, hydrogen cyanide and ammonia even under tebuconazole stress. Generally, tebuconazole at the recommended, two and three times the recommended field rate adversely affected the growth, symbiosis, grain yield and nutrient uptake in greengram in a concentration dependent manner. In contrast, the P. aeruginosa strain PS1 along with tebuconazole significantly, increased the growth parameters of the greengram plants. The inoculant strain PS1 increased appreciably root nitrogen, shoot nitrogen, root phosphorus, shoot phosphorus, and seed yield of greengram plants at all tested concentrations of tebuconazole when compared to the uninoculated plants treated with tebuconazole. The results suggested that the P. aeruginosa strain PS1, exhibiting novel plant growth regulating physiological features, can be applied as an eco-friendly and plant growth catalyzing bio-inoculant to ameliorate the performance of greengram in fungicide stressed soils.     

Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosa water network hospital population: invasion and selection of clonal complexes

Aims: To investigate infra-specific spatio-temporal dynamics of a hospital water network Pseudomonas aeruginosa population. To infer the origin of water network isolates and assess their potential health hazard. Methods and Results: 168 P. aeruginosa strains were isolated from tap waters and swabs of tap nozzle aerators of a hospital unit, over 2 years, and from rectal swabs and nosocomial infections. Genetic diversity among this collection was assessed by pulsed field gel electrophoresis of SpeI restricted genomic DNA. Virulence gene sets, biofilm properties, and hypochlorite resistance were analysed. Exactly 68% of the water samples and 74% of the tap nozzle aerators harboured P. aeruginosa. The strains were divided into 22 clonal lineages, with one dominant clone shown to have been involved in a nosocomial infection. Conclusions: An important turnover among the P. aeruginosa hospital population was observed. Some clonal lineages were found to persist, spread in the unit, and diversify into clonal complexes. Rectal carriage appeared an important source of contamination of the water network. Significance and Impact of the Study: High P. aeruginosa infra-specific population diversity suggested a broad ability in colonizing water networks but persistence analysis indicated a strong selection leading to the emergence of dominant clones.     

Isolation of toxic metal-tolerant bacteria from soil and examination of their bioaugmentation potentiality by pot studies in cadmium- and lead-contaminated soil

Heavy metals, also regarded as toxic metals, are the important environmental pollutants that affect all forms of life. Accumulation of toxic metals in plants results in various biochemical, physiological and structural disturbances, leading to inhibited growth and sometimes plant death. Toxic metal contamination disturbs the soil ecology as well as the agricultural productivity. Several indigenous microbes can withstand the effect of toxic metal and play a vital role in the revival of tarnished soil. In the present study, soil samples were collected from contaminated crop field of Cachar district of Assam, India. Segregation, enumeration and identification of bacteria from soil samples were performed. Among all the tested isolates, very few were able to withstand a high concentration of Cd and Pb in nutrient agar plates. Toxic metal-tolerant bacteria were identified as Pseudomonas aeruginosa and Bacillus cereus. The isolates having a higher tolerance for Cd and Pb were taken into consideration for pot studies. P. aeruginosa strain SN4 and strain SN5 showed significant results at Cd- and Pb-contaminated soil, evidenced by the healthy growth of Oryza sativa seedlings. However, B. cereus strain SN6 showed high tolerance towards Cd and Pb, but pot experimental studies showed adverse effects on seedling germination and shoot growth of O. sativa. P. aeruginosa strains were significantly able to reduce the negative impact of Cd and Pb in the soil, thus finding an alternative in removal, recovery and remediation of toxic metal-contaminated crop field.     

Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.     

Effect of hypochlorous acid solution on the eradication and prevention of Pseudomonas aeruginosa infection,serum biochemical variables,and cecum microbiota in rats

In this study, hypochlorous acid solution, a weak acid, provided as drinking water to rats, was evaluated for its ability to eradicate and prevent Pseudomonas aeruginosa infection, while monitoring its simultaneous effect on serum biochemical variables and microbiota in the rat cecum. The results suggest that the solution could not eliminate the bacteria in the experimentally infected rats; however, the administration of a 10-parts-per-million (ppm) hypochlorous acid solution as drinking water was effective in inhibiting horizontal spread of P. aeruginosa infection among cage mates. Additionally, exposure to hypochlorous solution did not have any effect on serum biochemical variables of the rat including levels of total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total bilirubin, lipase, amylase, urea nitrogen, total protein, calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), except for potassium (K) levels. The most frequently isolated bacteria in the rat cecum included species belonging to Bacteroidales, Lactobacillus, Clostridiales, Erysipelotrichaceae, Akkermansia, Coriobacteriales, and Firmicutes. The ratio of the terminal restriction fragment length polymorphism (T-RFLP) peaks did not differ across rats administered with 5 and 10 ppm weak acid solution as compared to the control group for any of the bacteria, except for Erysipelotrichaceae and Firmicutes, where the ratio of T-RFLP peaks was higher in the 5 ppm group for Erysipelotrichaceae and in the 10 ppm group for Firmicutes than that in the control group (P<0.01). The results suggest that the weak acid hypochlorous solution could not eradicate P. aeruginosa completely from rats. The solution was effective in preventing infection without affecting serum biochemical variables; however, some of bacterial microbiota may have changed due to administration of the solution.