Introduction
Background
Respiratory tract infections (RTIs) are common viral and bacterial infections that affect the lungs, throat, sinuses, and airways. The identification of these pathogens is necessary for specific and effective treatment of RTIs [1]. Usually, the identification of pathogenic organisms is cumbersome as they are harmful and adherence to strict precautionary steps in handling and confirming their identity is required. Equipment used in handling pathogens ought to be sterilised to avoid contaminating surfaces and exposing people to the risk of infections [1, 2]. Together with proper sterilisation procedures, wearing appropriate protective clothing is a precautionary measure that effectively reduces the risk of exposure to pathogens.
As microorganisms are pervasive, they are always interacting with humans and become part of everyday life. From this viewpoint, it is important to identity microorganisms for their profiles are critical in ascertaining their impact on humans, other animals, and the environment [2]. Ample evidence shows that bacteria are pathogens that can cause serious diseases amongst humans and animals, while others are important because they are used in beneficial production systems such as fermentation. The process of identifying unknown bacteria normally starts with their morphological features, followed by staining, biochemical reactions, and susceptibility tests. Genome sequencing is an advanced method that can be applied as a confirmatory test. In general, the bacterial nomenclature has no basis on genetic relatedness but on physical traits and metabolic aspects [2, 3], which comprise a set of unique traits making it possible to differentiate between bacteria.
Significant Infections of the Respiratory Tract
RTIs refer to infections affecting both the lower and upper respiratory tract. Comparatively, the upper RTIs are less pathogenic than the lower RTIs. Numerous RTIs-related deaths occur due to infections of the lower respiratory tract. Common infections of the lower respiratory tract include bronchitis, influenza, and pneumonia [4, 5]. Evidence shows pneumonia as the most common and serious form of RTIs despite the fact that influenza affects both the upper and lower respiratory tracts.
Normally, RTIs that affect the throat, sinuses, lungs, and airways occur due to viral infections and, to some extent, bacterial infections too. In humans, children are more susceptible to RTIs because their immune system is weaker than that of adults [1]. Nonetheless, the elderly are also prone to RTIs due to their advanced age [2]. It is, therefore, important to have a comprehensive understanding of RTIs because these infections spread in a variety of ways. Airborne matter bearing the viruses or bacteria has the capability of travelling far and spreading infections to large numbers of people. Direct and indirect contact with contaminated surfaces also contributes to the spread of the infections [2]. Proper hygiene practices are encouraged to minimise the proliferation and spread of RTIs. The use of a handkerchief in covering the nose and mouth during sneezing is one of the simplest ways of decreasing airborne and contact spread of microorganisms in sputum.
Though most RTIs subside without any treatment, taking painkillers, drinking plenty of water, and having ample rest effectively relieve symptoms. Antibiotics are discouraged unless a causative organism of RTIs has been established [1]. Since prevention is more appropriate than treatment, the observation of good hygiene is effective in avoiding and controlling the spread of RTIs [2]. However, in case the RTIs are serious, one ought to take over-the-counter medications such as antibiotics or painkillers. Otherwise, symptoms of RTIs mostly ease off after one to two weeks without the use of medication.
Microbiological Investigations on the Elderly
Generally, medical-related tests provide an accurate diagnosis of RTIs and offer reliable methods of drawing conclusions. Respiratory diseases among the old are among the main causes of morbidity and mortality. These tests are important among the old because symptoms can overlap due to viral and bacterial pathogens and cause inaccurate diagnosis and prognosis [2, 3]. Subsequently, opportunistic infections take advantage of RTIs and complicate their diagnosis and treatment. RTIs are prevalent among the elderly due to the existence of chronic conditions and deterioration of health. The elderly have underlying medical conditions that be complicated by infections such as RTIs. In addition, the deterioration of health with age reduces the immunity and increases the risk of bacterial or viral infections.
Benefits of Investigating the Respiratory Tract
RTIs are deadly infections to vulnerable groups, especially children and the elderly. Tests to establish the integrity of the respiratory system are important in ensuring the health of these vulnerable groups. The expectation is that specimens collected from the elderly have viral or bacterial pathogens [2], which have the potential of causing RTIs. Since RTIs are common, people often seek medications and most of the time they take medications without diagnosing their conditions. Acute sinusitis and bronchitis are some of the most common cases of RTIs that contribute to the abuse of antibiotics [3]. The general recommendations are that people should visit a physician when they present symptoms of RTIs, particularly when they appear serious. It is important for people, especially the elderly, with pre-existing medical conditions such as heart disease, diabetes, lung or kidney conditions, or on steroid medication, to seek medication even with the slightest signs of RTIs [2, 4]. Therefore, one should not overlook the symptoms of RTIs under such conditions since the mildest form of the condition can have detrimental effects.
The aim of the laboratory report was to explore the basic tests that form the foundation of microbiological methods employed in identifying unknown bacteria species. The tests revolve around a clinical sample obtained from an elderly person in intensive care. The patient seems to have underlying factors that have worsened his health condition. To understand the nature of RTIs, the specimen was tested to identify pathogenic microbes that are responsible for the condition. Tests indicated opportunistic infections that exploited the weak immune system of the elderly under investigation.
Materials and Methods
The sputum specimen (coded 7) was gram stained on horse blood agar (HBA), chocolate agar (CHA), and MacConkey agar (MCA), and then it was incubated for 24 hours at 370C. The identification process started with the observation of lactose fermentation [3] on the MacConkey media. Biochemical tests such as catalase and oxidase reactions were done following Mosby’s Manual of Diagnostic and Laboratory Tests [6]. The catalase test ascertained the organism’s ability to carry out a reduction reaction that led to the colour change and production of gas. MacConkey agar was used to establish fastidiousness of the organism.
Subsequently, the nitrate reduction test and growth on nutrient agar (NA) at 420C tests were done, according to the protocol in Textbook of Diagnostic Microbiology [7], and then used to identify the organism in this test. Susceptibility tests were done according to the protocol in Susceptibility Methods: Dilution and Disk Diffusion Methods [8] on Mueller Hinton Agar (MHA). The antibiotics used included Gentamicin, Ciprofloxacin, Imipenem, and Ceftazidime. The bacterium was cultured on plates containing each of the antibiotics and the inhibited region was observed. The responses of bacteria in terms of growth were recorded as either sensitive or not.
Results
The gram staining results revealed the presence of gram-negative bacilli and polymorphs. However, the bacteria were visible as rod-shaped bacteria in the MacConkey growth media. On the other hand, the HBA and CHA media had large and heavy growths as the colonies had metallic sheens and the characteristic smell of Pseudomonas. Though the MCA media had good growth, no lactose fermentation was observed.
Table 1: Summary of Biochemical Tests on Specimen 7.
Legend: (+) positive test (-) negative test.
Biochemical tests (Table 1) indicate the presence of gram-negative bacilli that was either Pseudomonas or Vibrio. The nitrate reduction test was positive for there was gas production. After zinc was added, the absence of a red/pink colour change was positive for nitrate reduction. The growth at 420C on nutrient agar was positive with dense growth observed. The susceptibility test indicated fluorescent pigment visible on the MHA agar. Figure 1 shows the workflow that led to the identification of RTIs in the sputum.
Table 2: Susceptibility Test Results.
Legend: S = sensitive.
Therefore, the susceptibility test confirms that the organism was Pseudomonas aerugenosa.
Discussion
MacConkey Agar is a selective growth medium exclusively used for detecting gram-negative bacteria. MacConkey Agar has been formulated to suppress the growth of gram-positive bacteria. When Specimen 7 was streaked on the media, rod-shaped microorganisms were seen on MacConkey [3]. Further, MacConkey has both selective and differential properties where the bacteria ferment mannitol and release acidic by-products that either promote or inhibit further growth of the bacteria, as the acidic concentrations build up. Although the organism was incapable of fermenting mannitol, growth on the MacConkey agar was indicative of the presence of fastidious gram-negative bacteria in the specimen.
HBA is for the cultivation of non-fastidious and fastidious microorganisms with the indication that haemolysis took place in the bacterial cultures. Chocolate agar is non-selective, enriched, and crucial for the isolation of pathogenic organisms. The media is appropriate for culturing fastidious respiratory organisms. The growth characteristics observed in Specimen 7 meets the criteria for respiratory bacteria [6, 7]. HBA is a nonspecific media, which does not distinguish between gram-negative and gram-positive organisms. A selective media for either kind of bacteria would have indicated if organisms in the specimen were gram-negative or gram-positive [5]. Whereas CHA confirmed the organism to be fastidious, HBA revealed it as a non-electrolytic one. The varying levels of specificities in these two media proved important in distinguishing microorganisms in the sputum and pinpointing the pathogenic one as Pseudomonas. Vibrio carries out haemolytic reactions on HBA but not Pseudomonas.
As the bacteria produced catalase that broke down hydrogen peroxide [9], the organism was sensitive to antibiotics in all the susceptibility tests done. Other biochemical tests, such as the oxidase test, were positive and exhibited a colour change. In the test, the presence of cytochrome oxidase in bacteria was ascertained. The enzyme facilitates the transportation of electrons between donors and a redox component, tetramethyl-p-phenylene diamine dihydrochloride, which is reduced to a deep purple colour [9, 10]. The organism being studied produces catalase and is oxidative (Table 1). These two tests are characteristic of Pseudomonas and Vibrio organisms that are mostly screened using this procedure.
A nitrate test determines the ability of organisms to reduce nitrate (NO3) to nitrite (NO2). This reaction is dependent on the production of a nitrate reductase enzyme by the organism [6]. The nitrate reduction test was positive with the production of gas. After zinc was added, the absence of a red/pink colour change was positive for nitrate reduction. In this case, the colour change did not occur for there was no reduction of nitrate. The enzyme had already performed denitrification, leading to the formation of ammonia or nitrogen. Thus, the test confirmed the presence of aerugenosa in the specimen.
The specimen colour (light green mucoid sputum) was indicative of the presence of aerugenosa. In part, the sample was obtained from an elderly man in the intensive care unit with pneumonia. Literature indicates that the bacterium is a common cause of gram-negative infections in patients who have a weak immune system, such as the old and hospitalised patients [1, 9, 10, and 11]. The medical conditions of the patient are therefore suggestive of Pseudomonas but not Vibrio.
Antibiotic susceptibility tests done involved the use of Gentamicin CN10, Ciprofloxacin CIP5, Imipenem IMP10, and Ceftazidime CAZ30 to determine the organism’s effectiveness. The testing on organism treatment options followed the Kirby-Bauer method [9]. The minimum and maximum zones of inhibition were recorded at 18 and 38 for Gentamicin and Ciprofloxacin, respectively (Table 2). The sensitivity test proved that Ciprofloxacin had the minimum effect against the organism, whereas Gentamicin had the maximum inhibitory effect, and hence a suitable drug against the organism.
The bacterium, aeruginosa, causes infections that have been considered a great threat due to the high mortality rate recorded for both pneumonia and bacteraemia conditions. Recently, the organism has developed resistance to antibacterial drugs. The bacteria have been documented to be highly adaptable because of its big genome [1, 9]. The adaptation has made the bacteria to be a multi-drug resistance (MDR) organism [10]. The elderly are prone to pseudomonal infection because of underlying factors such as diabetes, lung diseases, and the presence of invasive devices. Age is also a considerable factor that has increased the occurrence of MDR organisms [10, 11]. Such predisposing health conditions compromise the immune system and render the elderly vulnerable to opportunistic diseases and microorganisms. The rule of the thumb is to undertake comprehensive medical exams on elderly patients to establish their vulnerabilities.
The symptoms associated with aerugenosa infection in the bloodstream include malaise, chills, body aches, faintness, nausea, diarrhoea, and declined urination [1, 10]. In the case of pneumonia, symptoms include fever and chills, difficulty in breathing, cough and bloody mucus that can be yellow or green. The incubation period of the bacteria is usually between 24 and 72 hours. The complications associated with the infection are life-threatening and range from compromising the host immune system, with constant pneumonia attacks, urinary tract infections, and bacteraemia [10]. Perhaps the greatest challenge with monitoring and evaluating RTIs is their overlapping symptoms with other medical conditions. Normally, lack of adequate medical skills to diagnose and the existence of overlapping medical conditions make patients overlook or take a long time before seeking medical services. Studies have found that most people prefer to take easily dispensable medication for conditions that might be more serious [1, 9, and 11]. The development of multi-drug resistance in most pathogens perhaps arises from the patients’ use of antibiotics in an unwarranted manner.
As an opportunistic pathogen, aeruginosa exists in healthy individuals without necessarily causing harm. Pathogenesis of this bacterium is dependent on a complex group of factors because of its toxigenic and invasive attributes. The bacterium attaches to a surface and colonises it [1, 10], causing infection at the point of attachment [9, 10], then multiplies and spreads in the bloodstream, causing systemic infection [11]. Elucidation of the infection mechanism shows that attachment on the cell surface is crucial for systemic infection to occur. The attachment step increases virulence at one point before the pathogen spreads to the rest of the body through the bloodstream.
Although aerugenosa is increasingly becoming resistant to drugs, a combination of drugs is effective in minimising its effects on the host [10]. Evidence shows that a combination of penicillin and aminoglycoside are effective in the elimination of the bacterium [9, 10, and 11]. For antimicrobial susceptibility tests, results indicated that Gentamicin is the most effective and specific for aerugenosa [9]. The use of a two-drug regimen is subject to physician’s advice. The use of a combination of beta-lactams and aminoglycosides such as gentamicin, amikacin, and tobramycin are desirable for patients who have been on a single drug regimen.
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