Incidence of Pathogens and Their Drug Susceptibility Pattern in Blood and Urine Culture
INTRODUCTION
1.1 Background
Blood and Urine are the most commonly received specimen in routine microbiology laboratories, with many thousands of antimicrobial sensitivity results being issued each day (Barrett et al., 1999). The bacteriological examination of the urine is must for the diagnosis of urinary tract infection. Bacterial bloodstream infections are a leading cause of morbidity and mortality worldwide. In the United States approximately 200,000 patients develop bacteramia of fungaemia annually with estimated associated mortality ranges of 20 of 50% and septicaemia is the 13th leading cause of death. In addition, antimicrobial resistance in some of the most frequent bacterial species isolated from blood such as Staphylococcus aureus or Streptococcus pneumoniae has reached worrying levels (Decousser W.J, 2003). The urine and blood specimen is easy to obtain and can be collected in several different ways. A quantitative culture result can help diagnose significant bacteriuria and is performed by most laboratories.
Microorganisms are causative agents of many diseases. The identification of the bacteria causing the disease is often essential for the life and wellbeing of a patient. Blood borne pathogens are bacteria, viruses and parasites found in human blood and other body fluids. They can infect and cause disease in humans (Fatima Hamadi1 et at., 2008). Two pathogens recently receiving the greatest attention are the Hepatitis B virus (HBV) and Human Immunodeficiency Virus (HIV). Other pathogens which can also be of concern are Herpes, Meningitis, Tuberculosis, Epstein-Barr Virus, Lyme Disease, Malaria, and Syphilis, to name a few (Roy K.R., 2001). In healthy persons, properly obtained blood specimens are sterile. Although microorganisms from the normal respiratory and gastrointestinal flora occasionally enter the blood, they are rapidly removed by the reticuloendothetial system.
Blood culture is the single most important procedure to detect systemic infection due to bacteria. If a blood culture yields microorganisms, this fact is of great clinical significance provided the contamination should be excluded. Urine secreted in the kidney is sterile unless the kidney is infected. Uncontaminated bladder urine is also normally sterile. The urethra, however, contains a normal flora, so that normal voided urine contains small numbers of bacteria. Because it is necessary to distinguish contamination organisms form etiologically important organisms, only quantitative urine examination can yield meaningful results (Jawetz, 2001).
The wide variability of clinical symptoms and the ongoing difficulties concerning the rapid and specific laboratory diagnosis, contribute to the fact the sepsis primarily remains a clinical diagnosis. To contribute to a more tailored antibiotic coverage of the patient early in the course of the disease, modern diagnostic concepts favour the qualitative and quantitative molecular biological detection of blood stream pathogens directly from whole blood. This offers a very attractive alternative to the currently applied less sensitive and much more time-consuming blood culture-base laboratory methods. Moreover, recent study results suggest an increasing impact of molecular detection methods with short turn-around times for more effective treatment and better outcomes of patients with sepsis and septic shock. In the short term, such tests will not substitute conventional blood culture despite their superior rapidity and sensitivity, mainly because of higher cost. The amazing speed of ongoing scientific developments means, however, that techniques that might appear complicated, labour intensive and costly today, will develop to become the future standards in the microbiological diagnosis of patients with sepsis and septic shock. (Barrett p., et. Al., 1999).
In this context, the current study was designed to isolate and identify the etiological agents of blood and urine infections. Besides these, antimicrobial resistance and sensitivity pattern of the isolate was also determined to find out suitable prophylactic agent to treat the above mentioned infections.
1.2 Literature review
1.2.1 Urinary tract infections
The anatomical structure of the mammalian urinary system is such that the external genitalia and the lower aspects of the urethra are normally contaminated with a diverse population of microorganisms. The tissues and organs that compose the remainder of the urinary system, the bladder, ureters, and kidneys are sterile and therefore urine that passes through these structures is also sterile. When pathogens gain access to this system, they can establish infection (Cappuccino, 1996). Urinary tract infections (UTIs) are some of the most common infections experienced by humans, exceeded in frequency among ambulatory patients only by respiratory and gastrointestinal infections. It is also the most common cause of nosocomial infections in adults. E.coli is the leading pathogen and it was significantly more predominant in bacteremia Urinary tract infection (UTI) than non bacteremia UTI. Escherichia coli is the commonest cause of community and nosocomial urinary tract infection (UTI). Urinary tract infection (UTI) is a broad term that encompasses both asymptomtic microbial colonization of the urine and symtomic infection with microbial invasion and inflammation of urinary tract structures. Apart from the outer one-third of the female urethra, the urinary tract is normally sterile. From a microbiologic perspective, urinary tract infection exists when pathogenic microorganisms are detected in the urine, urethra,, bladder, kidney, or prostate. In most instances, growth of more than 105 organisms per milliliter from a properly collected midstream “clean-catch” urine sample indicates infection. The kids of infections that ESBL-producing E.coli can cause range from urinary tract infection, to – at the more serious end of the spectrum-case where they enter the bloodstream and cause blood poisoning. Infections with ESBL- producing E.coli are most common amongst the elderly, or those who have recently been in hospital or received antibiotic treatment. ESBL-producing E.coli are extremely rare in simple cystitis (Kenechukwu et al., 2000).
1.2.2 Circulatory system and blood infection
The main components of the human circulatory system are the heart, the blood, and the blood vessels. An average adult contains five to six quarts (roughly 4.7 to 5.7 liters) of blood, which consists of plasma, red blood cells, white blood cells, and platelets. Normally blood is sterile, bacteria occur transiently in the blood steam. Bacteremia may be a phase in the natural course of some infections such as typhoid fever and brucellosis and meningococcal infection, it also occurs as a spill-over effect in a serious infection when the patient’s defenses become inadequate, as in severe pneumonia or an extended soft tissue infection. Bacterial bloodstream infections are a leading cause of morbidity and mortality worldwide. (Alferd et al., 2005). Blood culturing is the “gold standard” for diagnosis of bloodstream infections (BSI). The identification of bacteremia and fungemia by culturing blood remains one of the most important roles of the microbiology laboratory. Most infections are caused by many different microbes, so it’s important to figure out which of them is causing the infection. A blood culture can be performed to determine whether the infectious agent is in bloodstream. Symptoms of an infection in bloodstream might include a high fever or chills. Blood culture procedure must design to overcome the intermittency and low order of magnitude bacteremias and fungemias and inhibit any antimicrobial properties or components of the blood. Among the seven variables affecting yields, the volume of blood cultured appears to be most important (Washington et al., 2000). Septicaemia denotes as overwhelming invasion of the blood steam from a focus of infection. The distinction between bactaemia and septicaemia is essentially clinical but there is a quantitative implication. Thus septicaemia is thought of as a life-threatening emergency that must be dealt with urgently (Andrew et al.,1989).
1.2.3 Microorganisms associated with blood and urine
Human microflora has been shown to contain microorganisms which refer to bacteria, microscopic fungi and protozoa. Some of them are intracellular parasites. Unfortunately, currently available methods are not sufficiently rapid and universal for slow growing bacteria, anaerobes, nonfermenters and other extraordinary microbes. Bacteria, the most familia of infectious agents, cause 90 percent of hospitalized infections in developing countries, although they compete with viruses for being the most diversified. For the most part, bacteria are the smallest free-living organisms in nature, having all the genetic material necessary to live independently. This is not to say that many of them don’t enjoy off other organisms, but as a class they are complete life forms unto themselves. Bacteria cause most of the serious, short-term infections we get and can be stopped with medications called antibiotics (Cappuccino, 1996).
A sample of urine from a patient with a suspected urinary treat infection is the most common type of specimen received by most clinical microbiological laboratories. The commonest condition of UTI is cystitis, due to infection of the bladder with a uropathogenic bacterium, which most frequently is Escherichia coli but sometimes Staphylococcus saprophyticus or especially in hospital-acquired infection, Klebsiella spp, Proteus mirabilis, other coliforms Pseudomonus aeruginosa or Enterococcus faecalls. Candida infection may occur in diabetic and immunocompromised patients. Rare infection organisms including Sreptococcus agalactiae, Steprococcus milleri, other, Streptococci, anaerobic Streptococci and Gardnerella vaginalis (Collius et al., 1986). More serious bacterial infections are acute phelitis and pyelonephuritis, in which the symptoms usually include join pain and fever and accompanied by a bactaeremia detectable by blood culture. The causative organism may be any of those that cause cystitis but, Staphylococcus aureus is responsible for some the cases (Stamm et al., 1980). Some bacterial and fungal agents of urinary tract diseases are illustrated bellow.
Organisms other than bacteria may also act as etiological agents of urogenital infection. Trichomonas vaginalis, a pathogenic flagellated protazoa is coomonly found in the vagina and under appropriate condition it is responsible for a severe inflammatory vaginitis. Candida albicans, pathogenic yeast is normally found in low numbers in intestines.
| Bacteria |
| Gram Negative |
| Gram Positive |
| Escherichia coli
Psendomonas aerugnosa Proteus uvlgsris Klebisella pneumoniae |
| Streptococcus aureus
Streptococcus pyotenes |
| Enterococci |
| Streptococcus faecalis
Streptococcus faecium |
| Figure 1.1: Bacterial agents of urinary tract diseases |
| Coccidioides immitis |
| Fungi |
| Blastomyces dermatitidis |
| Candida albicans |
Figure 1.2: Fungal agents of urinary tract disease (Cappuccino, 1996).
1.2.3.1 Escherichia coli
Escherichia coli (E.coli) is a bacterium that commonly lives in the intestines of people and animals. Most of the E.coli are normal inhabitants of the small intestine and colon and are non-pathogenic, meaning they do not cause disease in the intestines. Nevertheless, these non-pathogenic E.coli can cause disease if they spread outside of the intestines, for example, into the urinary tract (Where they cause bladder or kidney infections) or into the blood stream (sepsis) (Mitchell E., et al 2000).
1.2.3.2 Acinetobacter baumannii
Acinetobacter baumannii is a nonfermentive, aerobic, opportunistic, gram-negative coccobacillary rod. Morphological findings vary according to the phase of cell growth and exposure to antimicrobial agents. Acinetobacter baumannii commonly isolated from the hospital environment and hospitalized patients. This organism is often cultured from hospitalized patients sputum or respiratory secretions, wounds, and urine. In a hospital setting, Acinetobacter commonly colonizes irrigating solutions and intravenous solutions. Acinetobacter species have low virulence but are capable of causing infection. Most Acinetobacter isolates recovered from hospitalized patients, particularly those recovered from respiratory secretions and urine, represent colonization rather than infection (Shih1 M J 2007).
1.2.3.3 Staphylococci
Staphylococci are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes. Staphylococcus aureus form a fairly large yellow colony on rich medium; S.epidermidis has a relatively small white colony. S. aureus is often hemolytic on blood agar; S. epidermidis is non hemolytic. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. The bacteria are catalase-positive and oxidase-negative. S.aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. Nearly all Strains of S.aureus produce the enzyme coagulate; nearly all strains of . lack this enzyme. S.aureus should always be considered a potential pathogen; most strains of S.epidermidis are nonpathogenic and may even play a protective role in their host as normal flora. Staphylococcus epidermidis may be a pathogen in the hospital environment (Bae. T. 2004).
1.2.3.4 Candida
Candida is yeast and the most common cause of opportunistic mycoses worldwide. It is also a frequent colonizer of human skin and mucous membranes. Candida is a member of normal flora of skin, mouth, vagina, and stool. As well as being a pathogen and a colonizer, it is found in the environment, particularly on leaves, flowers, water, and soil. Infections caused by Candida spp. Are in general referred to as candidiasis. The clinical spectrum of candidiasis is extremely divers. (Jo-Anne H et al., 2001). Candidacies may be superficial and local or deep-seated and disseminated. Disseminated infections arise from hematogenous spread from the primarily infected locus. Candida albicans is the most pathogenic and most commonly encountered species among all (Abi-Sai et al 1997).
1.2.3.5 Salmonella typhi
Salmonella typhi is part of the Enterobacteriaceae family; it is a Gram-negative motile, aerobic rod which is faculttively anaerobic and there is serological identification of somatic and flagellar antigen (Reveendran R et al. 2007).
1.2.4 Antibiotics
Antibiotics are used for treatment or prevention of bacterial infection. Antibiotics may be informally defined as the subgroup of anti-infective that are derived from bacterial sources and are used to tract bacterial infections (M.K et al 1898). Some antibiotics are bactericidal (kill bacteria), others are bacteriostatic (arrest bacterial growth). Some antibiotics have a broad spectrum of activity, being active against a wide range of pathogens; others have a narrow spectrum of action. Streptomycin was the first major antibiotic to be discovered after penicillin. Many of the newer penicillins, such as ampicillin, such as ampicillin, possess a much broader spectrum of activity, some of them being active against Gram negative bacteria. Despite some adverse reactions in the human, effective antibiotics have been developed that have one or more of these modes of action on the bacterial cell:
A. Inhibition of cell wall synthesis
B. Alternation of cell membranes
C. Inhibition of protein synthesis
D. Inhibition of nucleic synthesis
E. Antimetabolic activity or competitive antagonism (Warren J.W, 1999).
1.2.4.1 Treatment of urine infection:
The major organisms causing UTIs are the Coliforms, Staphylococcus aureus and Proteus spp. Antibiotics which have been recommended to treat UTIs includes Ampicillin, Trimethoprim-Sulfamethoxazole, Flouroquinolones and Nitrofurantion. However due to incessant abuse and misuse of these antibiotics, extensive resistance of micro-organisms to these antibiotics has developed. The usual treatment for both simple and complicated urinary tract infections is antibiotics. The type of antibiotic and duration of treatment depend on the circumstances (Mezue Kenechulwu et al, 2004).
For patients with troublesome dysuria, phenazopyridine may help control symptoms until the antibiotics do (usually within 48 h). Ceftriaxone 125 mg IM plus either azithromycin or a fluoroquinolone is given for 10 to 14 day. For non-STD urethritis in men, trimethoprim-sulfamethoxazole or a fluoroquinolone is given for 10 to 14 days; women are treated with a regimen for cystitis asymptomatic bacteriuria in pregnant women is actively sought and treated as a symptomatic UTI, although many antibiotics cannot be safely used. Orar b- lactams, sulfonamides, and nitrofurantoin are considered safe in early pregnancy, but sulfonamides should be avoided near parturition because of a possible role in the development of kernicterus. Common regimens include ampicillin plus, gentamicin and a Fluoroquinolone, and broad-spectrum cephalosporins (eg, ceftriaxone, Aztreonam, b lactam/b-lactam inhibitor, combinations (ampicillin-sulbactam) and imipenem-cilastatin clavulanate, piperacillin-tazobactam ticarcilli are generally reserved for patients with more complicated pyelonephritis (Mayo Clinic, 2007).
1.2.4.2 Treatment of blood infections
Identifying the specific causative agent ultimately determines how sepsis is treated. However, time is of the essence, so a broad-spectrum antibiotic or multiple antibiotics will be administered until blood cultures reveal the cause and treatment can be made specific to the organism. Intravenous antibiotic therapy is usually necessary and is administered in the hospital. A number of different types of medications are used in treating sepsis. They include: Antibiotics, vasopressors, activated protein and others. Treatment with antibiotics begins immediately – even before the infectious agent is identified. The antibiotics are administered intravenously (IV). People with severe sepsis usually receive supportive care including intravenous fluids and oxygen (Mayo Clinic, 2007).
1.2.4.3 Trends of antibiotic resistance in Bangladesh
All antibiotics cause risk of overgrowth by non-susceptible bacteria. Excessive or inappropriate use may promote growth of resistant pathogens. Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome and a rate of about 1 in 108 per chromosomal replication. Some common pathogens are now resistant to antibiotics previously used frequently for treatment. Some strains of Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa are resistant to almost every antibiotic available. Uropathogens are resistance to commonly used antibiotics is causing concern in several other countries. Although rates of resistance to antibiotic in Bangladesh is about 60%. In Bangladesh, a resistance rate 18% to ciprofloxacin has also been reported and in Spain the resistance rate to norfloxacin is 13%.
1.3 Aims and Objectives
Probably 10% or fewer of asymptomatic bacteriuria patients develop renal failure attributable to the infection; hypertension is even rare. Chronic urinary tract infection is eradicate by short-term therapy (2-6 weeks) in about 25-35% of patients. Some of the others have relapses caused by the same organism; some have re-infection caused by other organisms. Again, infection in blood is very much fatal and can cause meningitis and other serious complications. So, antibiotic should be advised more carefully by keeping in mind the after effect of antibiotic resistance.
The present study had following aims and objectives:
a) To isolate and identify the causative agents of urine infections.
b) To isolate and identify the causative agents of blood infections.
c) To determine the antibiotic sensitivity and resistance pattern of isolated pathogens.
d) To find out the most suitable antimicrobial agents for treatment of urine and blood infections.
MATERIALS AND METHODS
2.1 Collection site and Number of specimens
The blood and urine samples were collected from the out-patients and admitted patients of United Hospital Limited, Gulshan, Dhaka from 10 October 2011 to 31 December 2011. A total 250 clinical specimens of blood and urine were cultured for isolation and identification of aerobic bacteria and antimicrobial susceptibility testing was also carried out.
2.2 Specimen collection and handling
2.2.1 Collection of blood samples
Venous blood samples were aseptically collected into blood culture bottles using sterile needles and syringes. The blood culture bottles utilized in this study were the BD BACTED PLUS Aerobic/ F and Anaerobic/ F (Becton Dickinson and Company). 10 ml of venous blood were collected from adult patients 50 ml blood culture bottles, while 5 ml were taken in the case of children.
Fig 2.1: Blood culture vial.
2.2.2 Collection of urine samples
In case of urine sample collection, patients were usually asked to submit mid-stream urine samples about 10-20 ml for analysis and culture, preferable the first urine excreted by the patient early in the morning in the commercial urine bottles.
2.3 Inoculation of the specimens
The bottles with specimens were immediately placed in the automated BACTEC 9120 incubator system. This system incubates specimens at 350 C with continuous agitation and uses a fluorescent technology to detect the quantity and rate of CO2 production (indicative of microbial growth) in every 10 min. Blood culture bottles were removed from the incubator after the automated system after determining that they were positive. After the incubation period, they were inoculated onto MacConkey agar and blood agar and incubated at 370 for 24.48 hours. The urine samples were immediately inoculated onto MacConkey agar, blood agar, and then the plates were incubated at 370 C for 24.48 hours. Each sample was plated onto 5% sheep blood agar and MacConkey agar using a calibrated 100p, delivering 0.01 ml of the sample. This was incubated at 370 C overnight and the observation was made the next day.
For the culture of blood and urine, 4 types of media were used:
- Blood agar for isolation of pathogenic bacteria,
- MacConkey agar for isolation of Enterobacterioceae,
- SDA agar for culture the fungal strains, and
- Mueller-Hinton agar medium for antibiotic susceptibility test.
2.4 Microscopic examination of urine
The microscopic examination of urine sample was performed as a wet film of uncentrifuged urine to determine whether polymorphs (pus cells) are present in numbers which indicated the infection in the urinary tract.
2.5 Identification of isolates
Following the incubation, colonies from the different media were characterized and identified using standard microbiological and biochemical scheme. The tests included gram and spore test, catalase test and oxidase test. Coagulase test was done for conformation of Staphylococcus spp in the catalase test positive.
2.5.1 Catalase test
The demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide. A small amount of the culture to be tested was picked from the agar plate with a clean sterile platinum loop and mixed with 3% hydrogen peroxide solution. The production of bubbles from the surface of the culture indicated the positive reaction.
2.5.2 Tube test/ coagulase test
To peform the test one coloni was emulsified with a small amount of citrate-treated plasma, and incubate at 370 C. The reaction is slow and usually requires overnight incubation. Less-virulent Staphylococci do not produce coagulase and are often collectively referred to as ‘coagulase-negative’. If the test is positive the plasma solidifies into a solid plug. To visualize this, tip the tube and watch the meniscus – if negative, it will remain horizontal; if positive it will rotate with the tube.
2.5.3 Oxidase test
The Oxidase test depends on the presence in bacteria of certain oxidases the transport of electron between electron donor in the bacteria and a redox dye – tetramethyle-p-phenylene-diamine.
2.5.4 MICROGEN TEST (A biochemical identification system for the common Enterobacteriaceae)
The Microgen GN – ID system employs 12 (GN A) standardized biochemical substance in micro wells to identify the family. Enterobacteriacease and other non-fastidious negative bacteria (oxidase negative and positive). The kit is intended laboratory use only.
2.5.4.1 Principle of the test:
The Microgen GN – ID system comprises to separate micro well test strips GN A and GN B. Each micro well test strips contains 12 standardize biochemical substrates which have been selected to the basis of extensive computer analysis. Dehydrated substrates in each well are reconstituted with a saline suspension of the organism to be identified. If the individual substrates are metabolized by the organisms, a colour change occurs during incubation or after addition of specific reagents. The permutation of metabolized substrates can be interred interoperated using the microgen identification system software (MID-60) identify the test organism. The GN A micro well test strip is intended for the identification of oxidase negative, nitrate positive glucose fementers comprising the most commonly occurring genera of the family Enterobacteriaceae. The GNA and GNB micro well test strips are used together to produced a 24 substrate system to identify non-fastidious gram negative bacilli (oxidase negative and positive) in addition to all currently recognized species of the family Enterobacteriaceae.
Figure 2.2: Inoculation of culture in micro wells of microgen test
2.5.4.2 Identification of isolates:
On the Microgen GN-ID A+B report from, the substrates have been organized into triples with each substrate assigned a numerical value (1, 2, 3 or 4). The sum of the positive reactions for each triplet forms a single digit or the Microgen Identification System Software, which generates a report of the five most likely organisms in the selected database.
Table 2.1: List of microorganisms that could be identified by microgen test.
| Acinetobacter baumannii | Salmonella typhi | Enterobacter aerogenes | Shigella boydii (GroupC) |
| Morganella morganii | Enterobacter agglomerans | Salmonella Group IV | Serratia rubidaea |
| Acinetobacter lwoffii | Salmonella cholerae-suis | Salmonella Group V | Yersinia enterocolitica |
| Proteus mirabilis | Enterobacter gergoviae | Salmonella Group I | Klebsiella pneumoniae |
| Acinetobacter haemolyticus | Salmonella Paratyphi A | Salmonella Group Illa | Serratia Marcescens |
| Proteus vulgaris | Enterobacter sakazakii | Salmonella Group II | Serratia liquefaciens |
| Citrobacter freundii | Salmonella gallinarum | Enterobacter cloacae | Klebsiella oxytoca |
| Providencia rettgeri | Escherichia coli | Shigella sonnei (Group D) | Klebsiella ozaenae |
| Citrobacter diversus | Salmonella pullorum | Hafnia alvei | Klebsiella rhinoscleromatis |
| Providencia stuartii | Escherichia coli-Inactive | Providencia alcalifaciens | Shigella flexneri (Group B) |
| Edwardsiella tarda | Shigella dysenteriae | Salmonella Group VI |
2.6 Antibiotics susceptibility test by Disk Diffusion method: (the Kirby Bauer technique)
Mueller-Hinton agar was considered to be the best for routine susceptibility testing which was done by Kirby-Bauer technique.
2.6.1 Inoculation of the Mueller-Hinton agar plate with test organism:
The isolated colony from the various media was inoculated on the Mueller-Hinton agar plate by the spreading technique.
2.6.2 Application of Discs to Inoculated Agar Plates:
1. The antimicrobial discs were dispensed onto the surface of the inoculated agar plate. Each disc must be pressed down to ensure complete contact with the agar surface. Whether the discs were placed individually or with a dispensing apparatus, they must be distributed evenly so that they are no closer than 24 mm plate or more than 5 discs on a 100 mm plate. Because some of the drug diffuses almost instantaneously, a disc should not be relocated once it has come into contact with the agar surface.
2. The plates were inverted and placed in an incubator set to 350C for over night.
The antibiotic disk potency and the specific organism for the specific antibiotic are:
Table 2.2 Antibiotic potency of the various antibiotics used in the study
| Antibiotic | Antibiotic potency (?g) | Organism |
| Ampicillin | 10 | Gram positive |
| Azithromycin | 15 | Gram positive |
| Amoxyclve | 30 | Gram positive |
| Vancomycin | 30 | Gram positive |
| Penicillin G | 10 Units | Gram positive |
| Doxycylin | 30 | Gram positive |
| Gentamincin | 10 | Gram positive |
| Imipenem | 10 | Gram positive |
| Amikacin | 30 | Gram positive |
| Amoxycilin | 10 | Gram positive |
| Azithronam | 30 | Gram positive |
| Ceftazidime | 30 | Gram positive |
| Ceftriaxone | 30 | Gram positive |
| Ciprofloxacin | 5 | Gram positive |
| Morepenem | 10 | Gram positive |
| Cotrimoxazole | 25 | Gram positive |
| Imipenem | 10 | Gram positive |
| Nalidixic Acid | 30 | Gram positive |
| Nitrofurantion | 300 | Gram positive |
Zone sizes were measured from the edge of disc to the zone which is given in the following table.
Table 2.3 Interpretation of antibiotic susceptibility
| Zone size | Interpretation |
| 1. Equal to wider than or not more than 3 mm smaller than the control | Susceptible |
| 2. Zone size greater than 3 mm, but smaller than the control by more than 3 mm | Intermediate |
| 3. Zone sizes 3 mm or less | Resistant |
RESULTS
3.1 The number of patients, age and sex distribution
The total numbers of 250 patients were studied in this research work, and among them 111 were female and 139 were male. 115 blood and 135 urine samples were collected from the patients and the age range was 0 to 90 years.
Table 3.1 the age and sex distributions of the patients
| Age range-0 to 90 years | |||
| Sex | Type of Specimen | No. of Sample | No. of Positive Culture |
| Male | Blood | 67 | 18(26.86%) |
| Urine | 72 | 30(41.66%) | |
| Female | Blood | 48 | 12(25%) |
| Urine | 63 | 17(26.98%) | |
Table 3.1 shows that out of 115 patients blood culture were positive in 30(26.08%) study cases, out of 67 male patients blood culture were positive in 18(26.86%) and out of 48 female patients blood culture were positive in 12(25%) patients. No significant difference was observed in between sex groups.
A total 30 strains of bacteria were isolated. Among them, A.baumannii 7(23.33%) most common predominantly islolated bacteria followed by S.paratyphi A 6(20.00%), S.typhi 5 (16.67%), E.coli and Pseudomonas sp. there were same 3(10.00%) in each bactria. Furthermore, Klebsiella sp. 2(6.67%), Candida sp. 2(6.67%), Enterobacter sp. 1(3.33%) and S.epidermidis 1(3.33%) were isolated.
Table 3.2: Prevalence of microorganism in specimen collected from male / female patients in blood culture
| Organisms | Numbers of identified organisms | Percentage | Total number |
| Acinetobacter baumannii | 7 | 23.33% | 30 |
| Pseudomons sp | 3 | 10% | |
| S.epidermidis | 1 | 3.33% | |
| Salmonella typhi | 5 | 16.67% | |
| Salnonella Paratyphi “A” | 6 | 20.00% | |
| Klebsiella sp | 2 | 6.67% | |
| Candida sp | 2 | 6.67% | |
| Enterobacter sp | 1 | 3.33% | |
| E.coli | 3 | 10% |
Table (3.1) shows that out of 135 patients urine culture were positive in 47 (34.81%) study cases. Out of 72 male patients urine culture were positive in 30(41.66%) and out of 63 female patients urine culture were positive in 17(26.98%) patients. No significant difference was observed in between sex groups.
Table 3.3: Prevalence of microorganism in specimen collected from male / female patients in urine culture
| Organisms | Numbers of identified organisms | Percentage | Total number |
| E.coli | 26 | 55.32% | 47 |
| Candida | 4 | 8.51% | |
| Acinetobacter baumunnii | 3 | 6.38% | |
| Pseudomonas sp. | 4 | 8.51% | |
| Beta haemolytic Streptococcus | 2 | 4.26% | |
| Non haemolytic Streptococcus | 3 | 6.38% | |
| Klebsiella sp | 4 | 8.51% | |
| Serratia sp | 1 | 2.13% |
A total 47 strains of bacteria were isolated. Among them E.coli 26 (55.32%) was the most common predominantly isolated bacteria followed by klebsiella sp., Pseudomonas sp., Furthermore, Acinetobacter baumannii, Non heamolytic Streptococcus 3(6.38%) each were isolated.
3.2 Pus cell count in urine sample
Counts of the pus cells were given in table 3.4
Table 3.4: Enumeration of pus cells in urine sample
| Sample number (n=22) | Pus cell count/H.P.F (high power field) | Age (years) | Sex |
| Sample 1 | 50-60 | 55Y | Male |
| Sample 2 | 30-40 | 69Y | Female |
| Sample 3 | 30-40 | 66Y | Female |
| Sample 4 | 20-30 | 58Y | Male |
| Sample 5 | 10-15 | 69Y | Female |
| Sample 6 | 8-10 | 40Y | Female |
| Sample 7 | 8-10 | 37Y | Female |
| Sample 8 | 5-8 | 42Y | Female |
| Sample 9 | 5-7 | 65Y | Female |
| Sample 10 | 3-5 | 26Y | Male |
| Sample 11 | 2-4 | 65Y | Male |
| Sample 12 | 2-4 | 45Y | Male |
| Sample 13 | 2-3 | 39Y | Female |
| Sample 14 | 2-3 | 53Y | Male |
| Sample 15 | 2-3 | 65Y | Female |
| Sample 16 | 2-3 | 47Y | Female |
| Sample 17 | 1-2 | 85Y | Male |
| Sample 18 | 1-2 | 20Y | Female |
| Sample 19 | 1-2 | 15Y | Female |
| Sample 20 | Uncountable | 33Y | Female |
| Sample 21 | Uncountable | 27Y | Female |
| Sample 22 | Uncountable | 31Y | Female |
3.3 Identification of isolates
3.3.1 Culture characteristics of different microbes
Cultural characteristics of isolated microbes are given in the table 3.3 and following figures.
Table 3.5: Colony morphology of different types of microorganisms on different media
| Microorganisms | Media | Colony characteristics |
| E.coli | MacConkey agar | Small, pink colony |
| Acinetobacter spp | Blood agar | Non-Lactose fermenting round shape cology |
| K. pneumonia | MacConkey agar | Mucoid, large pink colonies |
| Candida sp. | Saboroud dextrose agar | Mucoid, wet convex colonies |
| Enterobacter sp | Blood agar | Gray white small colony |
| Salmonella paratyphi | Blood agar | Gray white small colony |
| Pseudomonas sp. | MacConkey agar | Small transparent, colonies |
| Staphylococcus epidermidis | Blood agar | Large white colonies with zones of B heamolysis |
Figure 3.1 Culture of E.coli in MacConkey agar plate form urine sample.
Figure 3.2 Culture of Enterobacter in MacConkey agar plate from blood sample
Figure 3.3 Culture of Pseudomonas sp. in MacConkey agar plate
3.2.2 Gram staining
All the microorganisms were negative except S. epidermidis and S. aureus. Results of Gram staining of the isolates are given below.
Table 3.6: Results of Gram staining
| Microorganisms | Gram reaction |
| Pseudomonas sp. | Gram Negative |
| E.coli | Gram Negative |
| Enterobecter sp. | Gram Negative |
| Staphylococcus epidermidis | Gram Positive |
| Staphylococcus aureus | Gram Negative |
| Klebsiella sp. | Gram Negative |
| Acinetobacter baumannii | Gram Negative |
| Salmonella paratyphi and salmonella paratyphi “A” | Gram Negative |
Table 3.7 Results of oxidase tests, Catalase tests and coagulage tests
| Sample | Colony characteristic and media | Oxidase test | Catalase test | Coagulage test | Presumptive result |
| Urine | Small pink colony on MacConkey agar | – | – | E.coli | |
| Blood | Non-Lactose fermenting round shape colony | – | + | Acinetobacter baumonnii | |
| Blood | Non-Lactose fermenting transparent colony of MacConkey agar | – | + | Salmonella paratyphi A | |
| Blood | Non-Lactose fermenting transparent colony of MacConkey agar | – | + | Salmonella paratyphi B | |
| Blood and Urine | Large white colonies with zones of a heamolysis | + | + | – | S.epidermidis |
| Blood and Urine | Non-Lactose fermenting grape like colony | + | + | Pseudomonas sp. | |
| Blood | Mucoid, large pink colonies | – | + | Klebsiella pneumoneae | |
| Urine | Pin point colony on blood agar | – | – | Enterobacter sp. |
3.3 Results of Biochemical test (Microgen test)
After reading the result of Microgen GN ID A and B considering the microgen idenfication system software, the following organisms were identified (Table 3.6 and 3.7). The distribution of the isolates and their sources are presented in table.
The microorganisms identified in the blood were candida sp, E.coli, S.epidermidis, salmonella para typhi “A”. Enterobacter sp, Acinetobacter baumannii, salmonella typhi. Pseudomonous, Klebsiella sp. The most frequently occurring organism in blood samples was Acinetobacter baumannii (23.33%) followed by S. Paratyphi A (20.00%)
Figure 3.4: Interpretation and result of microgen tests
Out of the 47 positive cultures of urine, E.coli was most frequent (55.32%) and candida sp. (8.51%), Klebsiella sp. (8.51%), Pseudomonas sp. (8.51%) were the second highest in occurrence. And other organisms that were isolated from the urine specimens were beta haemolytic Streptococcus, Serratia sp, Acinetobacter sp, Non-haemolytic Streptococcus sp.
3.4 Antibiogram of isolates
The antibiogram of bacteria associated with the blood and urine of the patients in the hospital were reported here.
Figure 3.5: Drug sensitivity pattern of Pseudomonas spp. on Mueller-Hinton agar plate.
3.4.1 Sensitivity / Resistance of Antibiotics against Microorganisms of urine sample
The sensitivity patterns of organisms from urine sample were shown in the table 3.8 for E.coli, table 3.9 for Klebsiella sp. against the following antibiotics:
AMC=Amoxyclavonic acid, AK=Amikacin, CAZ=Ceftazidime, CRO=Ceftriaxone, CIP=Ciprofloxacin, MEM=Meropenem, IPM=Imipenem, CN=Gentamycin, CFM= Cefixime, FEP=Cefepime, NET= Netilmycin, SXT= Cotrimoxazole, F= Nitrofurantoin, NA= Nalidixic acid, PB= Polymyxin B, TZP= Tazobactam/Piperacillin
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Figure-3.6: Sensitivity pattern of E. coli against different types of antibiotics.
E.coli were sensitive (100%) to imipenem, Amikacin, Morepenem, Nitrofuration were 84.62% each sensitive. On the other hand, E.coli showed 96.15% resistance against tazobactam and followed by nalidixic acid (88.46%).
Figure–3.7: Sensitivity pattern of Klebsiella sp. against different types of antibiotics.
Klebsiella sp. Isolates in this study were found 75% sensitive to amiacin, imipenem, morepenem, tazobactum and 100% resistance to amoxyclave, ciprofloxacin, ceftraxone, ceftazidime, cefepime and gentamycin.
Non haemolytic Streptococcus showed 100% sensitivity to amoxyclave, nitrofuration, linezolid, vancomycin and 100% resistance to cefepime, cotrimoxazole, cefixime, nalidixc acid.
Sensitivity / Resistance of antibiotics against Microorganisms of blood sample
For the blood isolated organisms, the sensitivity and resistant patterns were shown in the table 3.10 for Acinetobacter baumannii, the table 3.11for E. coli, table 3.12 for S. paratyphi A, table 3.13 for Klebsiella sp. and table 3.14 for Pseudomonas sp. against the following antibiotics: AMC=Amoxyclavonic acid, AK=Amikacin, ATM=Aztreonam, AZM= Azithromycin, CAZ=Ceftazidime, CRO=Ceftriaxone, CIP=Ciprofloxacin, MEM=Meropenem, IPM=Imipenem, CN=Gentamycin, Do=Doxycycline, OX=Oxacillin, TZP=Piperacilline/Tazobactam, CFM= Cefixime, FEP=Cefepime, NET= Netilmycin, C=Chloramphenicol, SXT= Cotrimoxazole,
Figure-3.8: Sensitivity pattern of Acinetobacter baumannii against different types of antibiotics.
A.baumannii were 100% sensitive to cotrimozazole and 85.71% each sensitive to ciprofloxacin and imipenem. Resistances were found 100% against ceftazidime, gentamicin and netelmicin and 85.71% to amikacin, amoxyclavonic, cefepime and ceftriaxone.
Figure -3.9: Sensitivity pattern of S. paratyphi Aagainst different types of antibiotics.
Bacterial isolates of S.paratyphi A showed 100% sensitivity to ceftriaxone and cefepime each. Sensitivity to ciprofloxacin, cefixime, cotrimoxazole, chloramphenicol and Aztreonam were also 100% and resistance to ampicillin was 16.67%. Finally, 100% highly resistivity was found against Azithromycin.
S.typhi were 100% sensitive to ceftriaxone, ciprofloxacin, cefepime again, cefixime and Aztreonam were also showed 100% sensitivity followed by Azithromycin 80%. On the other hand, resistance were observed to cotrimoxazole, chloramphenicol and ampicillin 60% each.
Figure-3.10: Sensitivity pattern of E. coli against different types of antibiotics.
E.coli were sensitive (100%) to amikacin, amoxyclavonic acid, cefepime, ceftriaxone, ceftazidime, cefixime, imipenem, netilmycin wrer also showed 100% sensitivity. But were 66.7% resistant to cotrimoxazole followed by ciprofloxacin, gentamycin, Morepenem, Tazobactum 33.33% each..
Figure-3.11: Sensitivity pattern of Pseudomonas sp. against different types of antibiotics.
Isolates of Pseudomonas sp. were 100% sensitive to imipenem, morepenem and tazobactum but 100% each resistant to contrimoxazole and cefixime. Amikacin, cefepime, ciprofloxacin, ceftazidime, gentamycin and netelmycin showed 66.7% sensitivity each. However, 66.7% resistances were found against amoxyclave and ceftriaxone.
Figure -3.12: Sensitivity pattern of Klebsiella sp. against different types of antibiotics.
Klebsiella sp isolates in this study were found 100% sensitive to imipenem and meropenem, 50% to Amikacin, cefepime and ciprofloxacin. On the other hand, 100% resistance were observed against ceftriaxone, ceftazidime and cefixime, Klebsiella sp showed 50% resistance against contrimoxazole, netelmycin and tazobactum, 50% resistivity was also found against amoxyclavonic acid.
3.4.3 Identification of ESBL and MRSE group
There were eleven ESBL producing E.coli and two Klebsiella sp. identified from the urine. One was resistance to 10 antibiotics and another was resistance to 13 antibiotics. The name of the antibiotics against which these bacteria were resistant were presented table 3.8
Table 3.8: ESBL producing microorganisms and name of the antibiotics
| Number of resistant isolates (n=13) | Name of resistance antibiotics | Name of antibiotics |
| Ecoli (11) | AMC, FEP, CEP, CIP, SXT, CN, NET, CRO, CAZ, NA, F | 11 |
| Klebsiella sp. (2) | AMC, FEP, CEP, CIP, CN, NET, CRO, CAZ, NA, | 9 |
From the antibiotic sensitivity tests from blood samples one S. epidermidis was isolated that was MRSE. It showed resistance to 7 antibiotics. The name of the resistance antibiotics were given in the following table
Table: 3.9 MRSE group and the antibiotics
| Number of resistant isolates (n=1) | Name of resistance antibiotics | Name of antibiotics |
| 1 | AMP, DO, OX, CIP, SXT, CFM, E | 7 |
DISCUSSION
Blood and urine infection is very severe and immediate diagnosis is necessary to find out the causative agents, because both infections may become fatal if untreated. Sometimes patients use antimicrobial agents before diagnosis. But antimicrobial resistance is an issue of great significance for public health at the global level. Considered as wonder drugs, antibiotics are after prescribed inappropriately and inadequately and have thus become one of the highly abused agents. Bacterial pathogens causing acute infections are increasingly exhibiting resistance to the commonly used antibiotics and have become a great threat to public health. The increasing antibiotic resistance problems, largely due to widespread and irrational use of antimicrobial agents in hospitals and the community, is a cause of great concern, especially in developing countries (Lakshmi V., 2008). For this reason, this project work was regarding to the collection of two types of samples e.g. blood and urine and the microbiological study of the pathogens isolated from those samples. A total 250 specimens were collected from the indoor and outdoor patients of United Hospital Ltd. Dhaka. Among the total number of patients, blood samples were collected from 67 male and 48 female. Urine samples were collected from 72 male and 63 from female patients.
Firstly, urine was assessed under a high power field (HPF) for the presence of pus cells before microbiological analysis. Two urine samples collected from two female patients had uncountable pus cells which indicated severe urine infections. Some of the urine samples had 30-40, 50-60 pus cells in microscopic examination. That means pathogens caused severe damage in urinary tract so pus cells were released in urine enormously. Normally, there should be only an occasional red blood cell in the urine (2-3 per high power field). Hematuria, the presence of abnormal numbers of red blood cells in the urine may be due to: Glomerular disease, tumors that erode any part of the urinary tract, kidney trauma, renal infarcts, acute tubular necrosis, upper and lower urinary tract infections, traumatic catheterization, passage of renal stones. In male the normal range is 5-8 and in females it is up to 10 per high power field (HPF). All investigations are to be interpreted in the background of patient’s symptoms.
Pus cells don’t always mean infection (Shigemura K. et. al., 2005). Some of the urine samples that were containing pus cells showed no growth.
The more interesting aspect of this study was use of automatic system named Microgen test by which pathogenic microbes specially members of Enterobacteriocae family were identified my microngen identification system software. The automated system’s ability to enumerate the bacterial populations in the original clinical specimen attained a high degree of accuracy (Isenberg et al., 1979). 47 positive cultures were found from all the urine samples and it was found that Escherichia coli were the most frequent causative agents. E. coli is the most