Gram Stain Reaction Research Articles

PREVALENCE OF URINARY TRACT INFECTIONS AMONG FEMALES OF REPRODUCTIVE AGE GROUP IN TERTIARY CARE TEACHING HOSPITAL

Objective: The purpose of the current study is to isolate, characterize, and evaluate the antibiotic susceptibility profile of the pathogenic bacteria causing urinary tract infections (UTIs) in females of reproductive age. Methods: This is a cross-sectional descriptive study carried out in a Tertiary care teaching hospital for 1 year. A total of 100 urine samples from female patients in the reproductive age range of 15–44 years constitute the study’s sample size. Clean catch midstream urine samples were taken in sterile containers and inoculated on a different culture medium, such as cysteine, lactose, and electrolyte-deficient agar, to isolate the UTI pathogen. MacConkey agar was inoculated in parallel to target the growth of Gram-negative bacilli. Thereafter, using the common microbiological procedures, all positive cultures with significant bacteriuria were identified at the species level based on the colony features, Gram-staining reaction, and pattern of biochemical profiles. To investigate the pattern of antibiotic susceptibility, the disc diffusion method was used. Results: Among the 100 study cases, the prevalence of UTI was seen in 38% of individuals and UTI was absent in 62% of individuals. Among the females (38% cases) who had UTI, 81% had symptomatic UTI and 19% had asymptomatic UTI. Among the 81% symptomatic individuals of UTI, all they showed burning micturition followed by 74% of individuals showed dysuria Among the uropathogens isolated, majority were Escherichia coli (40.7%) followed by CONS (22.3%) Majority of the E. coli isolates showed sensitivity to piperacillin/tazobactam and nitrofurantoin. Conclusion: If left untreated, UTI is a significant public health issue. Early detection and timely treatment will lower the risk of developing further UTI complications, as well as the patient’s pain, length of stay in the hospital, and economic loss.

Isolation and Laboratory Diagnosis of Antibiotic-resistant E. coli from Surgical Wounds of Inpatients at Zagazig University Hospitals, Egypt

E. coli is one of the most common community and nosocomial pathogens and it mainly causes skin and soft tissue infections. So, the current study aimed to study the isolation of antibiotic-resistant E.coli strains in Egypt, Hospitals. Study their antibiotic susceptibility profile and therapy to control and present the transmission of E. coli strains among the health care settings.120 pus samples were collected from post-operation wounds of a study group of inpatients in Egypt, Hospitals. Bacteria colonizing the wounds were isolated on specific culture media thereafter purified and divided into 4 groups depending on their morphological characters and Gram's stain reaction Isolated bacteria were divided into 4 groups. Group (I) represented 40% of total isolates; while group (II) & (III) represented 25% and group (IV) 22%. Isolated bacteria related to groups I, II, III & IV were preliminary identified as S. aureus. E. coli, K. pneumoniae and P. aeruginosa, respectively. Antibiotic susceptibility of the 4 groups of isolated bacteria against 10 different antibiotics revealed that Ciprofloxacin showed the highest activity against E. coli. Data revealed that strain encoded E.coli 6 is a multi-resistant strain as it resisted 13 antibiotics out of 15 (86.7%).

Application of a Rapid Method for Gram Differentiation of Human Pathogenic and Non-Pathogenic Bacteria without Staining (Short Communication)

The Gram-stain reactions of pathogenic and non-pathogenic bacteria isolated from clinical samples were accurately characterized by mixing a concentrated droplet of cells with 3% potassium hydroxide (KOH). Compared to standard Gram-staining procedures, the KOH test was rapid, simple, and completely accurate. Forty-one isolates representing Bacilus, Corynebacterium, Escherichio, Haemophilus, Klebsiella, Neisseria, proteus, Pseudomonas, Salmonella, Shigellae, Staphylococcus, and Streptococcus species were tested. Bacteria cell were removed from an agar medium with toothpick placed on a glass slide with drop of 3% KOH with rapid circular agitation. With Gram-negative stains the suspension become viscous as revealed by a mucoid thread that formed when the toothpick was lifted. Gram-positive bacteria dispersed in to the drop and did not have this reaction. One isolate of Corynebacterium caused an increase in drop viscosity, but did not produce a mucoid thread.

Advanced diagnostic platforms reform the diagnosis of infections diseases

The development of diagnostic techniques played an important role in the prevention and treatment of infectious diseases.The traditional diagnostic techniques, such as gram stain, microscopy, culture, antigen/antibody detection and polymerase chain reaction, were widely used in the clinical diagnosis. But they couldn′t meet the demands of clinic and infectious diseases surveillance. Therefore, the point of care testing and multiplex testing based on advanced diagnostic platforms were developing rapidly, which might have the potential to change infectious diseases diagnostics.(Chin J Lab Med, 2017, 40: 11-13) Key words: Infection; Point-of-care systems; Diagnostic techniques and procedures

Prevalence of methicillin-resistant <i>staphylococcus aureus</i> among healthy residents of Ekosodin community in Benin-City, Nigeria

Purpose: To determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in apparently healthy residents of Ekosodin community, a peri-urban settlement, in Benin City, Nigeria.Method: Nasal swabs collected from 200 randomly selected individuals, aged between 16 and 38 years, were used in the study. Isolates from the swabs were aseptically collected and characterized using standard and established microbiological methods, which included growth and fermentation on mannitol salt agar (MSA), colonial morphology, Gram-staining reaction and biochemical tests. Antimicrobial susceptibility test was performed on Mueller-Hinton agar (MHA) by modified Kirby-Bauer disc diffusion method.Results: S. aureus was isolated in 49.5 % (n = 99) of the 200 nasal swabs. Among these isolates, 43 % were from male residents and 22.2 % (n = 22) were MRSA. The MRSA isolates indicated relatively high rate of resistance to penicillins, moderate resistance to erythromycin and cefuroxime, and leastresistance to gentamicin, streptomycin, ceftriaxone, ciprofloxacin and co-trimoxazole. There was no significant gender difference in terms of the colonization of S. aureus (p < 0.05).Conclusion: Many apparently healthy residents of Ekosodin community are nasal carriers of MSRA. The need for rational chemotherapy, routine detection and regular surveillance of MRSA to limit its spread and reduce treatment failures is vital.

Microorganisms isolated from surgical wounds infection and treatment with different natural products and antibiotics

Surgical site infections (SSIs) are common nosocomial infections in surgical patients resulting in significant increases in postoperative morbidity and mortality. This study aimed to isolate and identify bacteria that cause SSIs in Medical Research Institute, Alexandria University, Egypt, and compare their sensitivities to selected group of antibiotics and natural products. Isolates from 20 patients with SSIs were identified by culturing on blood and MacConkey agars, Gram staining and biochemical reactions. Most Gram-negative isolates (Pseudomonas aeruginosa, Klebsiella spp, Acinetobacter spp) were sensitive to amikacin, imipenem. Gram-positive isolates were Enterococcus faecalis which was sensitive to chloramphenicol, vancomycin, and Staphylococcus aureus which was sensitive to amikacin, imipenem, chloramphenicol. Gram-negative isolates were more sensitive to olive oil at 70 and 100% concentrations, and Acinetobacter spp were resistant at 30% concentration. Gram-positive E. faecalis isolates were most sensitive at 30% concentration. Garlic and oregano oils were more effective against most Gram-positive and negative isolates. E. faecalis was resistant to garlic oil at 30% concentration.Klebsiella spp were resistant to Nigella sativa oil at all concentrations, Acinetobacter sppwere resistant at 30% concentration, and P. aeruginosa was more sensitive at all concentrations than Gram-positive isolates. P. aeruginosa and S. aureus were most sensitive, while Klebsiella spp were resistant to commercial oils (olive, garlic, and chamomile). Olive leaf extract was more effective at four concentrations against all Gram-negative isolates. Gram-positive and -negative isolates were most sensitive to whey mixed with honey. This study refers to the possibility of using olive, garlic and oregano oils, olive leaf extract, and whey mixed with honey in treatment of SSIs. Key words: Isolated bacteria, surgical wounds, natural herbal products.

Incidence of urinary tract infections (UTI) among children and adolescents in Ile-Ife, Nigeria

This study was carried out in order to determine the incidence of urinary tract infection in children and adolescents, identify the uro-pathogens responsible for the infection and study the antibiotic sensitivity patterns of the uro-pathogens. Clean voided mid-stream urine samples were collected in sterile universal bottles from 301 children and adolescents between the ages of 5 and 18 years at the Obafemi Awolowo University Teaching Hospital complex (OAUTHC) Ile-Ife, Nigeria from December 2005 - July 2006. Culture plates with bacteria counts greater than or equal to 1 x 105 cfu-ml-1 were taken as positive, thus indicative of Urinary Tract Infection (UTI). The bacteria isolates were identified based on colony morphology characteristics, Gram stain reaction and biochemical tests using API 20E kits. The identified bacteria were then tested in vitro with standard antibiotics disc to determine their antibiotics sensitivity patterns. The result of this study shows that 36 (11.96%) of the 301 patients studies had UTI. Of the 124 females examined, 28 (22.4%) had positive urine culture while 8 (4.56%) of the 177 males had significant bacteriuria. A total of 36 bacterial isolates were obtained.Escherichia coli constituted the predominant organism and was responsible for (52.77%) of the cases of UTI. This was followed by Klebsiella sp. (25%), Proteus mirabilis (13.89%),Streptococcus faecalis (5.56%) and Pseudomonas aeruginosa (2.78%). The antibiotics sensitivity test revealed a high level of resistant to cotrimoxazole, amoxicillin and colistin as more than 60% of the isolates were resistance to these. This study highlights the presence of multi-resistance P. aeruginosa and poor compliance of the pathogens in vitro to antibiotics commonly used in treating UTI. It is therefore suggested that appropriate antimicrobials be administered to reduce the risk of multiply resistance organisms developing and avert ineffectiveness of antibiotics. Prompt therapeutic intervention is also essential to prevent cases of asymptomatic UTI from becoming symptomatic with resultant damage. Key words: UTI, uro-pathogens, antibiotic sensitivity.

Bacillus oleroniussp.nov., a member of the hindgut flora of the termiteReticulitermes santonensis(Feytaud)

A new rod-shaped endospore-forming bacterium is described, which was isolated from the hindgut of the termite Reticulitermes santonensis (Feytaud). The isolate stains Gram negative and its DNA has a guanine-plus-cytosine content of 35 mol%. Despite the Gram-staining reaction, both biochemical and physiological features place the isolate in the genus Bacillus and indicate a phenotypic resemblance to the Bacillus firmus-lentus group of species. On the basis of comparative 16S rRNA analysis and some phenotypic features the isolate clearly represents a new species for which the name Bacillus oleronius is proposed. The type strain is Bacillus oleronius Rt 10 (DSM 9356).

Evaluation of a scheme for predicting the gram-staining reaction of organisms causing bovine mastitis

Summary The accuracy of a scheme for predicting the gram-staining reaction of organisms causing bovine mastitis in cows with systemic signs of disease (anorexia) was evaluated over 1 year. Criteria for making the predictions included: season of year, stage of lactation, appearance of milk, detection and duration of teat injuries, and milk odor. It was possible to determine the cause by microbiologic culture of specimens from 136 of the 147 cows of the study. Of 78 infections caused by gram-negative (mostly coliform) organisms, 62 (79%) were predicted accurately to be caused by gram-negative organisms. Of 57 infections caused by gram-positive organisms, 45 (79%) were predicted correctly to be caused by gram-positive organisms. Correctly predicted as gram-positive organisms causing infection were: Actinomyces pyogenes in 20 of 21 (95%) cows; Staphylococcus sp in 14 of 22 (64%) cows; Streptococcus sp in 10 of 13 (77%) cows and Bacillus sp in 1 cow. Overall accuracy, in those instances when bacteria were isolated (136 cows), was 78%.

Biologic activity of type I and type II Fusobacterium nucleatum isolates from clinically characterized sites.

Fusobacterium nucleatum is a microorganism commonly cultured from periodontal disease sites. F. nucleatum isolates (120) were obtained from subgingival plaque samples taken from 27 clinically characterized sites using a selective culture medium. All isolates were verified by morphology, Gram-stain reactions, oxygen tolerance and biochemical reactions. A total of eight clinical isolates and two typed strains were used for further evaluation. In this study, there was no relationship found between GI and probing depth or between probing depth and frequency of isolation of Type I or Type II F. nucleatum colonies. There was a significant increase in isolation of Type II colonies with a GI of 2 (P less than 0.0001). All isolates tested shared lines of identity by double diffusion in agar and displayed similar ability to hemagglutinate sheep erythrocytes and a reduction in this hemagglutination activity by previous exposure to 50 mM D-galactose. All isolates tested showed similar protein patterns as determined by polyacrylamide gel electrophoresis. By the methods used, no differences were detected between Type I and Type II F. nucleatum; however, there was a statistically significant increase in Type II isolates with increasing levels of gingivitis.

The Inability of Pyrolysis Gas-Liquid Chromatography to Differentiate Selected Foodborne Bacteria

Pyrolysis gas-liquid chromatography (PGLC) and stepwise discriminant analysis (SDA) were ineffective when used to differentiate selected genera, species and strains of foodborne microorganisms. Each of 18 individual bacterial strains analyzed was grown, harvested and subjected to PGLC analysis. The resulting pyrolysis products were separated on a high resolution capillary column and the elution patterns (pyrograms) were subjected to stepwise discriminant analysis of 26 (a–z) characteristic peaks. Classification with the combination of PGLC and SDA was 87% accurate for gram-negative strains of bacteria and 94% accurate for gram-positive strains of bacteria. PGLC-stepwise discriminant· analysis correctly discriminated 80% of the bacterial strains according to the known gram-stain reactions. Only 63% were correctly classified to the genus level when all samples were compared. These findings point out the weak points for this method of bacterial analysis.

Superoxide Dismutase and Oxygen Metabolism inStreptococcus faecalisand Comparisons with Other Organisms

Streptococcus faecalis contains a single superoxide dismutase that has been purified to homogeneity with a 55% yield. This enzyme has a molecular weight of 45,000 and is composed of two subunits of equal size. It contains 1.3 atoms of manganese per molecule. Its amino acid composition was determined and is compared with that for the superoxide dismutases from Escherichia coli, Streptococcus mutans, and Mycobacterium lepraemurium. When used as an antigen in rabbits, the S. faecalis enzyme elicited the formation of a precipitating and inhibiting antibody. This antibody cross-reacted with the superoxide dismutase present in another strain of S. faecalis, but neither inhibited nor precipitated the superoxide dismutases in a wide range of other bacteria, including several other streptococci, such as S. pyogenes, S. pneumoniae, and S. lactis. The inhibiting antibody was used to suppress the superoxide dismutase activity present in cell extracts of S. faecalis and thus allow the demonstration that 17% of the total oxygen consumption by such extracts, in the presence of reduced nicotinamide adenine dinucleotide, was associated with the production of O(2) (-). A variety of bacterial species were surveyed for their content of superoxide dismutases. The iron-containing enzyme was distinguished from the manganese-containing enzyme through the use of H(2)O(2), which inactivates the former more readily than the latter. Some of the bacteria appeared to contain only the iron enzyme, others only the manganese enzyme, and still others both. Indeed, some had multiple, electrophoretically distinct superoxide dismutases in both categories. There was no discernible absolute relationship between the types of superoxide dismutases in a particular organism and their Gram-stain reaction.

Isolation of lipopolysaccharides and the effect of Polymyxin B on the outer membrane of Corynebacterium autotrophicum

Lipopolysaccharides (LPS) from Corynebacterium autotrophicum were isolated and analyzed. Autotrophically grown cells contained 2--5 mg of partly purified LPS per g dry weight of lyophilized cells. Serological cross reaction with Lipid A antigen of Salmonella minnesota confirmed the presence of LPS in C. autotrophicum. Electron microscopy of negatively stained Polymyxin B-treated cells showed formation of blebs on the Outer Membrane indicating an interaction of Polymyxin B specifically with LPS. Up to now, no Gram-positive organisms are known which contain any LPS. Thus, C. autotrophicum, though giving opposite results when the Gram-staining reaction was applied by several authors, has to be classified into the group of Gram-negative bacteria.

Role of Candida albicans in Granulomatous II. In Vivo Degradation of C. albicans in Hepatic Macrophages of Mice

Hepatic granulomas were induced in mice by injection of blastospores, cell walls, and glucan of Candida albicans. Granulomatous reactions in liver tissue initially multiplied but later decreased. A dose-response relationship was apparent with up to 3 mg of inoculum. Shortly after injection of C. albicans spores, fungal elements appeared in liver macrophages and were detectable in granuloma and Kupffer cells for 20 days. Gram-stain, periodic acid-Schiff (PAS), and immunofluorescence reactions soon vanished, and the organisms could no longer be seen. Glucan of C. albicans, which lacked PAS and immunofluorescence reactivity, proved active in initiation of granulomas. Degradation of phagocytized spores of C. albicans, reductions of cytoplasm, and cell wall deformation and collapse support the premise that loss of PAS and immunofluorescence reactivity was caused by enzymatic breakdown of candida cell wall mannan in macrophages. We conclude that C. albicans can induce granulomatous reactions in mouse liver when the glucan that forms the cell wall matrix in Candida persists in identifiable residues.

Gram-staining mechanism of superficial oral epithelial cells.

On the basis of cytochemical tests, the gram-staining reaction of superficial human oral epithelial cells, principally perinuclear in distribution, was found to be caused by certain undetermined moieties of cytoplasmic amino acids rather than by nucleic acids, a possibility suggested by its intracellular distribution.

Bactericidal effect of hyperbaric oxygen determined by direct exposure.

SummaryExposure of small inocula of microorganisms on the surface of blood agar medium to high pressure oxygen (HPO; 100% O2 at 3 atmospheres absolute) has provided a simple direct method for quantitating its antibacterial effect upon 102 strains from among 20 microbial species. The bactericidal effect was dependent upon the duration and intensity of exposure to HPO, and was independent of common taxonomical characteristics such as cellular morphology, gramstaining reaction, and a strict or facultative growth requirement for normobaric oxygen.

The microflora of raw bulk tank milk.

Milk samples from 141 farms were plated on standard plate count agar and the colony count determined after incubation for 48 hours at 32 °C. Twenty colonies were picked at random from plates containing between 30 and 300 colonies. The isolates were inoculated into litmus milk and subsequently characterized on the basis of shape, Gram-staining reaction, catalase production, lactose fermentation, and the ability to form spores.Certain general trends in the flora were observed. In milk of a colony count less than 2 × 104 per ml, micrococci were the dominant group of organisms, and as the colony count of the milk increased the percentage of micrococci decreased and the percentage of Gram-negative rods and streptococci usually increased. In spite of these general trends a study of the flora of individual samples showed that quite marked variations did occur.

The gram-staining reaction of disintegrated micro-organisms.

SUMMARY: Disintegration of yeast and Clostridium welchii gave cell envelopes which stained Gram-negative by a modified Jensen's Gram-staining procedure (procedure l), but stained in part Gram-positive by a modification of the Gram stain in which the films were dried after application of the iodine and before application of the decolorizing agent (90% v/v ethanol; procedure 2). Procedure 2 appeared to give a typical Gram reaction in that a series of organisms gave identical results when stained by the two procedures. Disintegrated Gram-negative organisms gave only Gram-negative material when stained by procedure 2. The Gram-positive material could be removed from the yeast envelopes by extraction at pH 8, but attempts to 'replate' the envelopes with the extract were unsuccessful. The results suggest that the Gram-staining reaction of yeast and C. welchii is due in part to a specific staining component and in part to a factor dependent upon the intact nature of the cells. The latter may be associated with the stability or orientation of the staining component or with some membrane effect. A number of workers have noted that when Gram-positive organisms are broken they become Gram-negative (Benians, 1912, 1919; Burke & Barnes, 1929). These results have been taken as support for the theory that the Gram-staining reaction is due to the presence of a membrane in Grampositive organisms which is impermeable either to the dye + iodine complex or. to .the decolorizing agent. This effect of breaking cells may be equally well explained as being due to the degradation or disaggregation of a Gram-positive complex when the cell membrane is ruptured. It is not certain that Grampositive organisms always become Gram-negative when broken. Thus Burke & Barnes (1929) observed certain Gram-positive structures in preparations of broken yeast cells, and Kemp (1931) showed that broken yeast cells showed Gram-positive structures when stained by a particular modification of the Gram stain. The results reported in the present paper show that although certain Grampositive organisms which had been disintegrated by shaking with glass balls stained Gram-negative by Jensen's modification of the Gram method, a change in this procedure as simple as just allowing the slide to dry before applying the decolorizing agent resulted in the distintegrated organism staining Grampositive. METHODS Organisms. The following were used : Saccharomyces cerevisiae (baker's yeast, Distillers Co., Ltd. DCL) ; Clostridium welchii ; Sarcina lutea ; Escherichia coli ; Axotobacter chroococcum (NCIB 8003) ; Caryophanon sp. (kindly supplied by Dr T. Gibson).. S. lutea, C. welchii and E. coli were grown at 37 in a Lemco

Mode of Action of Hypochlorite Sanitizers with and without Sodium Bromide

Summary Increases in bactericidal effectiveness of solutions of a hypochlorite sanitizer of high alkalinity with NaBr·2H 2 O added were related directly to the concentrations of NaBr·2H 2 O, with the effect of chlorine being negligible. Solutions containing constant concentrations of NaBr·2H 2 O and increasing concentrations of the alkaline hypochlorite sanitizer decreased in relative effectiveness with increasing concentrations of the sanitizer. This reduction in effectiveness appeared to be related to increases in pH of the solutions. The term active chlorine is proposed to express the per cent of the total available chlorine obtained by titration of a chlorine solution without acidification. The amount of active chlorine in a hypochlorite solution is related to the pH of the solution; consequently, to the bactericidal effectiveness and corrosiveness of the solution. Explanation is offered as to the reason why NaBr · 2H 2 O increases the effectiveness of hypochlorite solutions of high alkalinity. The merits of considering bacterial resistance to germicides on the basis of species, rather than on the basis of the gram-stain reaction, are discussed.

Nitrite production by heterotrophic bacteria.

SUMMARY : Heterotrophic bacteria were studied which produced nitrite in the presence of ammonia and in the absence of nitrate. A soil extract medium was prepared which allowed good growth as well as nitrite production in the absence of nitrate. Quantitative data are recorded showing that ammonia decreases as nitrite accumulates when four different cultures are grown in the soil medium. Resting cell studies add further evidence that some heterotrophic bacteria can convert ammonia to nitrite. A defined medium was prepared containing glucose or sodium acetate as the carbon source and NH,CI as the nitrogen source. This medium supported growth and nitrite production ; however, optimum conditions for growth were not established. Neither growth nor nitrite accumulation was as great in defined media as in soil-extract media. Results from defined media and from resting-cell studies rule out the possibility of any nitrate contamination. Early in the literature reports were published by Fremlin (1903, 1914, 1929), Makrinoff (1909), Cutler (1930), Cutler & Mukerji (1931), Cutler & Crump (1933), Crump (1935), Nechaeva (1947) and others suggesting the possibility of nitrification by heterotrophic bacteria. Recently Fisher, Fisher & Appleman (1952) and Hutton & ZoBell (1953) have added more data supporting these early observations. Isenberg et al. (1954) described a streptomycete which produces nitrite from urethane. This paper presents evidence that certain Gram-negative bacteria isolated from soil form nitrite in the presence of ammonia in defined and undefined media. Ammonia disappears as nitrite is produced. Special precautions have been taken to exclude nitrate from the media. METHODS Bacteria. Sixteen nitrite-producing cultures were obtained from soil by enrichment techniques. These were shown to be pure. The organisms were found to be Gram-negative short rods probably belonging to group 2 or 3 of Taylor & Lochhead's (1938) classification of soil bacteria. This classification, based on morphology and Gram-staining reactions of cultures grown on soil extract semi-solid agar, has eight large subdivisions, group 2 consisting of short Gram-negative rods and group 3 composed of short Gram-variable rods.