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Interaction of coagulase-negative Staphylococcus species with bovine mammary epithelial cells.

Three coagulase-negative Staphylococcus species (CNS) (Staphylococcus epidermidis, Staphylococcus xylosus and Staphylococcus hyicus), from the milk of cows with mastitis, were used to evaluate adherence to and internalization by bovine mammary epithelial cells, and to investigate involvement of host cell signal transduction and host cell cytoskeleton rearrangement on internalization of CNS. S. xylosus showed highest adherence and internalization values of the species evaluated. Host cell cytoskeleton polymerization and protein kinase (PK) phosphorylation were required for internalization of CNS. Both protein kinase C (PKC) and tyrosine kinase (TPK) pathways were involved, but internalization of S. xylosus occurred preferentially through epidermal growth factor TPK activity. S. epidermidis and S. hyicus seemed to exploit other TPK pathways. Results of this study showed that S. xylosus, S. hyicus and S. epidermidis were able to adhere and internalize bovine mammary cells in a process that appeared to be receptor(s) mediated and exploited host signal transduction and cytoskeleton to induce an uptake signal.

Animals↗

Peritoneal macrophages and opsonins: antibacterial defense in patients undergoing chronic peritoneal dialysis.

The antibacterial activity of phagocytic cells and opsonins in peritoneal dialysis effluents from 21 patients undergoing chronic peritoneal dialysis (CPD) was studied. Effluents contained an average of 12 x 10(6) cells per liter that were predominantly macrophages. Macrophages phagocytized and killed opsonized Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli as efficiently as did polymorphonuclear neutrophils (PMNs) from healthy donors. Macrophage chemiluminescence was one-third of that observed with donor PMNs. In the absence of opsonins, macrophages efficiently phagocytized and killed S. aureus by binding S. aureus cell wall protein A to macrophage surface IgG. Nine (43%) of 21 effluents failed to opsonize S. epidermidis, and none opsonized E. coli. When present, titers of S. epidermidis opsonins were 50- to 100-fold lower than that of normal serum. IgG and C3 concentrations in effluent reflected its opsonic capacity. Macrophages from patients undergoing CPD thus have intact phagocytic and bactericidal functions. However, the low level of opsonic molecules and inadequate numbers of macrophages in the peritoneal cavity may predispose patients undergoing CPD to peritonitis.

Ascitic Fluid↗

In vitro modulation of keratinocyte-derived interleukin-1 alpha (IL-1 alpha) and peripheral blood mononuclear cell-derived IL-1 beta release in response to cutaneous commensal microorganisms.

The ability of a range of skin commensal microorganisms to modulate interleukin-1 (IL-1) release by cultured human keratinocytes and peripheral blood mononuclear cells (PBMCs) was investigated by a combination of enzyme-linked immunosorbent assays and bioassays. Three fractions (formaldehyde-treated whole cells, culture supernatants, and cellular fractions) were prepared from Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, and Malassezia furfur serovar B. The levels of immunochemical IL-1 alpha released by cultured keratinocytes during coincubations with these microbial fractions ranged from 0 to 136 pg/ml and were maximal after 72 h. No microbial fraction consistently upregulated immunochemical IL-1 alpha release by freshly isolated keratinocytes from two donors and a transformed cell line, all of which produced the cytokine constitutively to various extents. Bioassays revealed that most of the IL-1 released was biologically inactive. In contrast, whole cells of formaldehyde-treated P. granulosum and S. epidermidis significantly stimulated release of IL-1 beta by PBMCs from three donors compared with the negative control (culture medium). Release was maximal at 24 h. Coincubation with intact cells of the yeast M. furfur significantly decreased levels of IL-1 beta below the values for the negative control by PBMCs from all three donors. There was good correlation between bioassay data and immunoassay data for IL-1 beta, and the depressive effect of M. furfur cells on cytokine production by all three cultures of PBMCs was mirrored in the levels of bioactive cytokine. This reduction in IL-1 beta release by PBMCs by M. furfur may provide an explanation why dermatoses thought to be caused by this yeast are essentially noninflammatory or only mildly inflammatory.

Humans↗

Sterility testing of platelet concentrates prepared from deliberately infected blood donations.

BACKGROUND: In general the bacterial count in freshly donated blood is low and even lower in the corresponding platelet concentrates (PCs). By use of flow cytometry (FACS) for sterility testing, the reliability of early versus later sampling times was evaluated. STUDY DESIGN AND METHODS: Blood donations were spiked with various numbers of Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae. The corresponding PCs were prepared by the buffy-coat method and stored at 22 degrees C. A 20-mL sample was collected from each PC directly after preparation and after 8 hours. Samples were stored at 35 degrees C. Sterility testing of both PCs and samples was by FACS analysis at different time points. RESULTS: All stored PCs were found positive by FACS analysis, with detection times ranging between 8 and 24 hours (K. pneumoniae, B. cereus), 8 and 91 hours (S. aureus), and 144 hours (S. epidermidis). In the samples incubated at 35 degrees C, bacteria were detected after 8 to 19 hours (K. pneumoniae, B. cereus), 8 to 67 hours (S. aureus), and 19 to 43 hours (S. epidermidis). Some of the samples did not contain bacteria. CONCLUSION: Detection times for slow-growing bacteria are significantly shortened when PC samples are incubated at 35 degrees C: the numbers of bacteria in freshly prepared PCs may, however, be so low that the samples drawn for sterility testing do not contain a single bacterium. Our results do not support a shortening of the 24-hour or greater sampling time recommended by the manufacturers of established test systems, because also for consistent detection by FACS, bacteria need to grow in the PCs to sufficient numbers.

Bacteria↗

Vitreous and aqueous penetration of orally administered gatifloxacin in humans.

OBJECTIVE: To investigate the penetration of gatifloxacin, a novel extended-spectrum fourth-generation fluoroquinolone antibiotic, into the vitreous and aqueous humor after oral administration. METHODS: A prospective, nonrandomized study of 24 consecutive patients scheduled for pars plana vitrectomy between September 2001 and May 2002 at the Cullen Eye Institute. Aqueous, vitreous, and serum samples were obtained and analyzed from 24 patients after administration of two 400-mg gatifloxacin tablets taken 12 hours apart before the operation. Assays were performed using high-performance liquid chromatography. RESULTS: Mean +/- SD gatifloxacin concentrations in serum (n = 23), vitreous (n = 23), and aqueous (n = 11) were 5.14 +/- 1.36 micro g/mL, 1.34 +/- 0.34 micro g/mL, and 1.08 +/- 0.54 micro g/mL respectively. Mean +/- SD sampling times after oral administration of the second gatifloxacin tablet for serum, vitreous, and aqueous were 3.2 +/- 1.0 hours, 4.0 +/- 1.0 hours, and 3.9 +/- 1.1 hours, respectively. The percentages of serum gatifloxacin concentration achieved in the vitreous and aqueous were 26.17% and 21.02%, respectively. Mean inhibitory vitreous and aqueous MIC(90 ) levels were achieved against many pathogens, including Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Propionibacterium acnes, Haemophilus influenzae, Escherichia coli, Bacillus cereus, Proteus mirabilis, and other organisms. CONCLUSIONS: Orally administered gatifloxacin achieves therapeutic levels in the noninflamed human eye, and the activity spectrum appropriately encompass the bacterial species most frequently involved in the various causes of endophthalmitis. Because of its broad-spectrum coverage, low MIC(90) levels for the organisms of concern, and good tolerability, gatifloxacin represents a major advance in the prophylaxis or treatment of postoperative, posttraumatic, and bleb-associated bacterial endophthalmitis.

Administration, Oral↗

Penetration pharmacokinetics of topically administered 0.5% moxifloxacin ophthalmic solution in human aqueous and vitreous.

OBJECTIVE: To investigate the penetration of 0.5% moxifloxacin hydrochloride into the aqueous and vitreous after topical administration in humans. METHODS: A prospective, nonrandomized study of 20 patients scheduled for vitrectomy surgery between September 1 and December 31, 2003. Aqueous and vitreous samples were obtained and analyzed after topical administration of 0.5% moxifloxacin hydrochloride, every 2 hours (q2h) or every 6 hours (q6h), for 3 days before surgery. Assays were performed using high-performance liquid chromatography. RESULTS: Mean +/- SD moxifloxacin concentrations in the q2h group for the aqueous (n = 9) and vitreous (n = 10) were 2.28 +/- 1.23 and 0.11 +/- 0.05 microg/mL, respectively. Mean +/- SD moxifloxacin concentrations in the q6h group for the aqueous (n = 10) and vitreous (n = 9) were 0.88 +/- 0.88 and 0.06 +/- 0.06 microg/mL, respectively. The minimum inhibitory concentration for 90% of isolates (MIC(90)) was far exceeded in the aqueous for a wide spectrum of key pathogens, whereas it was not exceeded in the vitreous for several organisms. However, the minimum inhibitory concentration for 50% of the isolates was exceeded in the q2h vitreous group for Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus cereus, and other gram-negative pathogens. CONCLUSIONS: The Endophthalmitis Vitrectomy Study revealed that 94.2% of isolates from postoperative endophthalmitis are gram-positive pathogens. Moxifloxacin has a spectrum of coverage that appropriately encompasses the most common organisms in endophthalmitis. The pharmacokinetic findings of this investigation show that relatively high aqueous levels can be achieved after topical administration. Further studies will help define the precise role of 0.5% moxifloxacin ophthalmic solution in the treatment of or prophylaxis against intraocular infections.

Administration, Topical↗

3-Methyl-3-sulfanylhexan-1-ol as a major descriptor for the human axilla-sweat odour profile.

This study sets out to redress the lack of knowledge in the area of volatile sulfur compounds (VSCs) in axillary sweat malodour. Sterile odourless underarm sweat (500 ml) was collected from 30 male volunteers after excessive sweating. Five strains of bacteria, Corynebacterium tuberculostearicum, Corynebacterium minutissimum, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Bacillus licheniformis, were isolated and characterised for their ability to generate an authentic axillary odour from the sweat material collected. As expected, all of the five bacterial strains produced strong sweat odours. Surprisingly, after extensive olfactive evaluation, the strain of Staphylococcus haemolyticus produced the most sulfury sweat character. This strain was then chosen as the change agent for the 500 ml of odourless underarm sweat collected. After bacterial incubation, the 500-ml sample was further processed for GC-olfactometry (GC-O), GC/MS analysis. GC-O of an extract free of organic acids provided three zones of interest. The first was chicken-sulfury, the second zone was onion-like, and the third zone was sweat, clary sage-like. From the third zone, a new impact molecule, (R)- or (S)-3-methyl-3-sulfanylhexan-1-ol, was isolated and identified by GC/MS, MD-GC, and GC AED (atomic emission detector). (S)-3-methyl-3-sulfanylhexan-1-ol was sniff-evaluated upon elution from a chiral GC column and was described as sweat and onion-like; its opposite enantiomer, (R)-3-methyl-3-sulfanylhexan-1-ol, was described as fruity and grapefruit-like. The (S)-form was found to be the major enantiomer (75%).

Adult↗

Central venous catheter-related sepsis in a cohort of 366 hospitalised patients.

Five hundred two central venous catheters inserted in 366 patients were evaluated prospectively over a one-year period to determine the frequency and risk factors associated with catheter-related sepsis. For study purposes, in cases in which catheter infection was suspected but the initial blood cultures were negative, the catheters were replaced by guidewire technique; otherwise, the catheters were routinely changed after 21 days by guidewire technique. A catheter-related infection was suspected in 190 cases (190/502, 38%). A diagnosis of catheter-related sepsis was established in 50 patients, which represents 10% of the total number of lines (502). Over a total of 6428 days of catheter use, the infection rate was 0.8 cases of sepsis per 100 catheter-days. Staphylococcus epidermidis, Staphylococcus aureus, and Candida spp. were the most frequently isolated aetiological agents of sepsis. On univariate analysis, six variables affecting the rate of catheter-related sepsis were identified: neutropenia for more than eight days (p < 0.001); AIDS (p < 0.001); haematological malignancy (p < 0.001); administration of total parenteral nutrition (p = 0.001); duration of site use (p = 0.04); and high APACHE II score (p = 0.04). The logistic regression analysis revealed that AIDS and haematological malignancies were independent risk factors of catheter-related sepsis. Catheter replacement over a guidewire was no more likely to be associated with sepsis than was percutaneous catheter insertion. In conclusion, although the incidence of established catheter infection is much lower than the incidence of suspected infection, in most cases of suspected infection it is wise to change the catheter with the guidewire technique and wait for culture of the tip, rather than to remove the catheter immediately. Such a policy may help reduce the number of unnecessary catheter removals.

APACHE↗

Post-antibiotic effect of the new streptogramin RP 59500.

The post-antibiotic effect (PAE) of RP 59500, a new streptogramin antibiotic, was determined for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, and Streptococcus pyogenes. A 30 min exposure of Staphylococcus aureus to 5 micrograms/ml of RP 59500 produced a PAE of 1.9-6.9 h, and a 60 min exposure of 2.5 micrograms/ml produced a PAE of 3.2-8 h. A 30 min exposure of 5 micrograms/ml of RP 59500 of coagulase-negative staphylococci produced a PAE of 2.5-7.5 h. PAEs of constitutively erythromycin-resistant staphylococcal isolates were shorter than were the PAEs of highly susceptible isolates. A 30 min exposure to 5 micrograms/ml of RP 59500 produced a PAE of 7.5-9.5 h for Streptococcus pneumoniae and a PAE of greater than 18 h for Streptococcus pyogenes. RP 59500 produced a longer PAE with Staphylococcus aureus than did vancomycin, oxacillin or erythromycin. These results suggest that RP 59500 may be administered less frequently than would be suggested by its half-life.

Clindamycin↗

Effect of ofloxacin on oral and gastrointestinal microflora in patients undergoing gastric surgery.

The effect of ofloxacin on the microflora in saliva, gastric juice, and feces was evaluated in 24 patients undergoing gastric surgery. A single peroral dose of 400 mg ofloxacin was given to each patient 2-4 h before surgery. The concentrations of ofloxacin in serum, saliva, gastric juice, and gastric mucosa tissue were assayed. Only Branhamella cocci were affected in the saliva. In the gastric juice, both the aerobic and anaerobic flora were suppressed on the day of surgery, but increased in number afterwards. There was a significant correlation between gastric pH and the number of microorganisms isolated in the gastric juice on day 0. In faeces, the aerobic and part of the anaerobic flora were suppressed by the administration of ofloxacin. Ofloxacin was well tolerated by the patients. Four of 24 patients developed postoperative complications. Microorganisms isolated from wound sepsis and intraabdominal sepsis (three patients) were Staphylococcus epidermidis, Staphylococcus aureus, hemolytic streptococci group A, Streptococcus sp. and Escherichia coli. All related infections occurred in patients with very low ofloxacin serum concentrations, probably due to impaired absorption. Oral antibiotic prophylaxis is not recommended in patients in whom impaired gastric emptying can be expected.

Adult↗

Comparative in vitro activity of norfloxacin against urinary tract pathogens.

The in vitro activity of norfloxacin (MK366) against 477 aerobic gram-negative and gram-positive clinical isolates was compared to that of nalidixic acid, nitrofurantoin, ampicillin, cephalexin, trimethoprim, sulfamethoxazole, and the combination trimethoprim-sulfamethoxazole. Norfloxacin was more active than the other agents against all gram-negative organisms tested. Minimal inhibitory concentrations (MICs) of Pseudomonas aeruginosa were less than or equal to 0.125-32 mg/l with 90% inhibited (MIC90) by 4 mg/l; MICs of the Enterobacteriaceae including Serratia marcescens were less than or equal to 0.125-8 mg/l with an MIC90 of less than or equal to 4 mg/l. There was also excellent activity against the gram-positive cocci including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus and the enterococci, with MICs less than or equal to 0.125-4 mg/l and an MIC90 less than or equal to 4 mg/l. Only 8 of 477 organisms were norfloxacin-resistant (MIC greater than or equal to 16 mg/l): 3 of 100 Pseudomonas aeruginosa, 3 of 10 Pseudomonas maltophilia and 2 of 15 Streptococcus bovis strains. In contrast, 97% of the gram-positive cocci and 49% of the gram-negative bacilli were nalidixic acid-resistant (MIC greater than or equal to 32 mg/l). Norfloxacin shows excellent activity against a wide range of bacteria and merits further study as a urinary antibacterial agent.

Anti-Bacterial Agents↗

Antibacterial activity of the fourth-generation fluoroquinolones gatifloxacin and moxifloxacin against ocular pathogens.

The ideal ophthalmic anti-infective exhibits broad-spectrum activity against gram-positive, gram-negative, and atypical bacterial species. These pathogens can cause potentially blinding infections such as keratitis and endophthalmitis, both of which are associated with ophthalmic surgery or traumatic injury. These infections often require aggressive antibacterial therapy, preferably with newer generations of antibiotics. In this study, minimal inhibitory concentration (MIC) values for gatifloxacin and moxifloxacin were determined in vitro against bacterial strains that were isolated from suspected cases of bacterial keratitis and endophthalmitis. The ocular isolates included 7 gram-positive, 4 gram-negative, and 3 atypical bacterial species. Gatifloxacin and moxifloxacin exhibited similar activity against 6 gram-positive organisms: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, and Enterococcus faecalis. MIC90 values for the drugs against these isolates ranged from 0.08 mg/mL to 0.57 mg/mL and were comparable to previously published values against isolates from patients with systemic infections. The MIC90 for gatifloxacin against Streptococcus viridans was 0.22 mg/mL compared with 0.73 mg/mL for moxifloxacin (P = .011). Among the gram-negative isolates, the mean MIC90 for gatifloxacin against Pseudomonas aeruginosa was 1.28 mg/mL compared with 2.60 mg/ mL for moxifloxacin (P = .023). MIC90 values for gatifloxacin against Klebsiella pneumoniae and Enterobacter aerogenes were one fourth to one fifth the values for moxifloxacin. For the atypicals, the MIC90 values for gatifloxacin against Nocardia asteroides and Mycobacterium chelonae were one fourth the corresponding values for moxifloxacin. Gatifloxacin demonstrated a broad spectrum of activity against several key ocular pathogens tested in this study and was at least as effective as moxifloxacin against these pathogens.

Anti-Bacterial Agents↗

Cell surface energy, contact angles and phase partition. III. Adhesion of bacterial cells to hydrophobic surfaces.

The densities of adhesion of Staphylococcus epidermidis, Staphylococcus aureus and Serratia marcescens to five types of plastics were studied in relation to interfacial free energies at the aqueous interfaces of both the bacteria and the plastics. The free energy of adhesion of bacteria to plastic in an aqueous medium is a linear function of partititon of the bacteria between the solid surface and the liquid phase. These results show that the thermodynamics of the partitioning of a suspended particle between two immiscible liquid phases also apply to partitioning between a liquid and a solid phase.

Bacterial Physiological Phenomena↗

A novel electrical method for the prevention of microbial colonization of intravascular cannulae.

An electrical device designed to prevent microbial attachment to plastic intravascular cannulae is described. The device produced an electrical current (10 microA) in both carbon-impregnated and 'Hydrocath' cannulae. In-vitro models were utilized to demonstrate that the current can block several mechanisms by which microorganisms gain initial access to intravascular devices, including extra- and intra-luminal routes. The electrical system prevented organisms from traversing along both the external and internal surfaces of the cannulae. Effective prevention of cannula colonization was demonstrated subsequent to challenge with Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The findings suggest that the electrical device may offer in-vivo cannula protection from microbial attachment and colonization, thereby reducing the potential for infection.

Bacterial Adhesion↗

Effects of atmosphere of incubation on recovery of bacteria and yeasts from blood cultures in Tryptic soy broth.

A comparison was made of the results of blood cultures between 1974 and 1981 in unvented and transiently vented bottles of Tryptic soy broth under vacuum with CO2. A total of 14,646 isolates were available for statistical analysis. Significantly more isolates of Staphylococcus epidermidis, Staphylococcus aureus, Bacillus, Escherichia, Pseudomonas, Klebsiella, Serratia, Acinetobacter, Alcaligenes, Neisseria, and Candida were recovered from the vented bottle. Significantly more isolates of Corynebacterium, Haemophilus, Flavobacterium, Moraxella, Bacteroidaceae, and Peptostreptococcus were recovered from the unvented bottle.

Aerobiosis↗

The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines.

The comparison of copolymers containing sulfobetaine or phosphobetaine moieties for use as potential biocompatible coatings has been investigated. Two statistical copolymers were produced by a free radical polymerisation technique, one based on a sulfobetaine and the other on a phosphobetaine, both with a silyl group component to allow thermal crosslinking after coating. PMMA and glass discs were dip-coated with the polymers and their properties were compared to the uncoated controls. Bacterial adhesion to these coated materials was assessed using Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa. Human macrophages and granulocytes were used to assess the adhesion and activation of inflammatory cells whilst mouse 3T3 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion. In all cases the polymer coatings reduced cell adhesion with respect to the base materials. The phosphobetaine-based copolymer coatings were shown to be markedly superior to the sulfobetaine-based copolymer coatings.

3T3 Cells↗

Metabolic activation of bladder procarcinogens, 2-aminofluorene, 4-aminobiphenyl, and benzidine by Pseudomonas aeruginosa and other human endogenous bacteria.

Pseudomonas aeruginosa, an opportunistic pathogen of the human urinary tract, and other selected human endogenous bacteria were investigated for metabolic activation of the bladder procarcinogens, 2-aminofluorene (2-AF), 4-aminobiphenyl (4-AB), and benzidine (Bz). The cell-free extracts of Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Staphylococcus saprophyticus, Klebsiella pneumoniae, and intestinal anaerobes, Bacteroides fragilis, Clostridium perfringens, and Eubacterium aerofaciens produced increased histidine revertant frequencies with the tester strain Salmonella typhimurium TA98 in the Ames Salmonella mutagenicity assay. In addition, the cell-free extracts of Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens each showed the presence of a cytochrome P450 absorption peak in the carbon monoxide (CO) difference spectrum. This was not demonstratable for the other bacteria. Our findings indicate that human endogenous bacteria, which are opportunistic pathogens of the urinary bladder, can metabolically activate the bladder procarcinogens 2-AF, 4-AB, and Bz into mutagens. The metabolic activation by Pseudomonas aeruginosa, Bacteroides fragilis, and Eubacterium aerofaciens is mediated by a cytochrome P450 enzyme. For those organisms that induced metabolic activation but did not show a P450 absorption peak with the cell-free extracts, other oxidative enzymes may be involved.

Aminobiphenyl Compounds↗

Recovery of blood-borne bacteria from human urine.

Recovery from the urine of organisms causing bacteraemia may depend on the bacterial species involved. The survival of the more common species of bacteria which cause bacteraemia was examined in human urine, serum and normal saline. All species survived well or grew in serum. Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus sanguis and group A streptococci were killed in all urine samples. The number of colony-forming units of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus and group B streptococci either remained the same or increased in the urine, while the numbers of Escherichia coli and Klebsiella pneumoniae increased rapidly. These data suggest that the observed differences in recovery from urine of these bacterial species that cause bacteraemia are related to the viability of the species in human urine.

Bacteria↗