Molecular Microbiology - Bacterial Blood Stream Infections - Case Study Example

CASE STUDIES: MOLECULAR MICROBIOLOGY Case 1. A 67 year old leukaemic patient developed bacteriaemia in hospital. It was suspected that his plasticcentral line catheter was colonized as indicated by the swelling at the point of entry in his skin. Blood culture showed Gram-positive cocci in clusters. Because intravenous catheters are commonly used in cancer chemotherapy and are prone to infection, they pose a major problem in the care of patients with cancer, such as, leukaemia. Some catheter-associated infections can be treated with antibiotics, while in others the catheter must be removed. The commoner intravenous catheter-related infections are exit-site infections, as in this case, often with erythema around the area where the line penetrates the skin. What microbiological diagnostic test would carry out to identify this organism Bacterial blood stream infections are common in this given scenario, and Staphylococci are the second most prevalent bacteria. However, a smear suggesting the staphylococci or Gram-positive cocci in clusters in blood culture as in here, is not sufficient for the diagnosis of true bacterial blood stream infection before the species is identifiable, since the most frequent of this species, Coagulase-negative staphylococci or CoNS usually habituate in the skin, and there is always a chance of contamination of the culture bottles during the venipuncture. In contrast, such an infection due to Staphylococcus aureus is virulent by its intrinsic nature, and isolation in one blood culture bottle is clearly diagnostic and is an indication of initiation of antibiotic therapy. Thus the therapeutic decision making is based on identification of the organism (Beekmann, S. E., Diekema, D. J. and Doern, D. J., 2005). The first test obviously would be to do a light microscopic examination. Direct microscopic examination may provide a rapid, presumptive report of Gram-positive cocci resembling staphylococci. Isolation of S. aureus should be performed using 5% blood agar following an incubation period of 18-24 h in air at 35-37 C. Staphylococcus aureus ferments mannitol, resulting in a change in the colour of the medium from pink to yellow. Colony morphology may be used by the experienced observer to define presumptive staphylococci. A Gram stain appearance of cocci in clusters and a positive catalase test provide rapid indicators of staphylococci. However, in order to be able to distinguish between Staphylococcus aureus and the remaining members of the staphylococcus species, other tests are necessary. For clinical microbiological purposes, two or three simple tests suffice. The coagulase test detects the production of coagulase by S. aureus. In this test, one colony is mixed with plasma, incubated at 37 C for 4 h and observed for clot formation. Samples that are negative at 4 h are incubated and observed again for clotting at 24 h. The slide agglutination test detects clumping factor (ClfA). This is performed by making a heavy homogenous suspension of cells in distilled water on a glass slide to which a drop of plasma is added. Within 10 s, the mixture is examined for presence of clumping (Chapin, K., and M. Musgnug, 2003). How would you differentiate the cocci in clusters from those in chain Cocci in clusters are named as staphylococci. All staphylococci have the ability to convert hydrogen peroxide into nontoxic H2O and O2. Both coagulase positive and negative staphylococci produce catalase. This test differentiates them from cocci in chain or streptococci, which cannot produce catalase and hence are catalase negative (Chapin, K., and M. Musgnug, 2003). What is the principle of DNase test and what is the identity of this organism and why Support your answer with microbiological diagnostic facts. DNase or deoxyribonuclease is an extracellular enzyme that can hydrolyze deoxyribonucleic acid to oligonucleotides. Several varieties of deoxyribonucleases are distinguished on the basis of antigenic properties, response to inhibitory substances, hydrolytic end products, and activity in the presence of Ca and Mg ions. Staphylococcus aureus produces a heat-resistant DNase or thermonuclease in contrast to the DNase produced by the other bacteria are heat sensitive. The DNase test detects the production of deoxyribonuclease. The test isolate is streaked onto solid agar containing DNA and incubated for 24 h. Hydrochloric acid is added to the surface of the plate that precipitates unhydrolysed DNA to produce a white opacity. A zone of clearing around the bacterial growth indicates the production of deoxyribonuclease that results in the hydrolysis of DNA. This occurs due to the fact that DNA is precipitated by hydrochloric acid, but the hydrolytic products are soluble in the acid. This test is useful for intra and extrageneric differentiation of Staphylococcus, Serratia, Pseudomonas, and Flavobacterium. Positive clear zone indicating positive enzyme action in presence of heat indicates Staphylococcus aureus, whereas negative test indicated by cloudy precipitate in the surrounding medium indicates Staphylococcus saprophyticus (Johnson, JR., Owens, K., Gajewski, A., and Kuskowski, MA., 2005). Case 2: 2. A young female patient with cystitis and pyelonephritis developed bacteriaemia. Blood culture results grew lactose fermenting bacteria on MacConkey Agar and that was Gram negative bacilli. What is the most common bacterial cause of cystitis and pyelonephritis Escherichia coli which generally originate from faeces or the periurethral flora are responsible for most urinary tract infections, including cystitis and pyelonephritis. Most episodes of cystitis and pyelonephritis are caused by extraintestinal pathogenic Escherichia coli strains. After colonising the periurethral area from the fecal area, these organisms may ascend the urinary tract from the urethral meatus and infect the bladder. In some cases, these organisms continue to ascend through the ureters and cause acute pyelonephritis, involving one or both kidneys. Patients with acute pyelonephritis present with a range of symptoms including fever, flank pain and bacteriuria with or without diaphoresis, rigors, groin or abdominal pain and nausea and vomiting. Abdominal tenderness of one or both kidneys may be elicited on examination. In a small proportion of cases, including those with pyelonephritis, the bacteria may spread beyond the urinary tract and enter the blood. Perhaps, this is the case in this clinical scenario. What are the cultural and biochemical characteristics of this organism The genus Escherichia includes motile and non-motile bacteria which belong to the family Enterobacteriaceae. Whilst most strains of E. coli grow well on a range of microbiological culture media, the growth and isolation of some pathogenic strains requires specific methodology. Rapid lactose fermentation is a key diagnostic feature of media used for the initial isolation or subsequent confirmation of E. coli. MacConkey agar and E. coli broth are used respectively for the initial isolation and confirmation of suspect E. coli. The presumptive identification of E. coli has been improved by the introduction of chromogenic media that provide better diagnostic characteristics mediated by specific enzyme activity which yields colonies of a distinct colour. Another common enzyme exploited in chromogenic media is beta-D-galactosidase that is responsible for lactose fermentation and common in E. coli. Lactose fermentation remains a useful diagnostic feature of media for the isolation of urinary pathogens which in this case is E. coli. Cysteine lactose electrolyte-deficient or CLED agar is used for routine diagnostic urinary bacteriology. This medium is recommended because it is reported that 1.5% of E. coli isolated from urine require cysteine and that subsequent broths used for their identification will require cysteine supplementation. The non-selective medium CLED supports the growth of a wide range of urinary pathogens whilst preventing swarming of Proteus spp. Lactose-fermenting organisms, including E. coli, lower the pH of the medium, which turns from green to yellow. Strains of E. coli are commonly distinguished from other faecal coliforms by their ability to grow and produce gas from lactose at 44 C and indole production from tryptophan. Thus those who ferment lactose slowly or not at all together with absent indole production and synthesis of lysine decarboxylase are not recognised as E. coli (Lloyd, AL., Rasko, DA., and Mobley, HLT., 2007). Give two examples each of the somatic O:H antigens of this organisms that are associated with cystitis and pyelonephritis. The correlation between specific O and H antigens with different pathotypes of E. coli has led to serotyping being used for identification purposes. The serotyping of E. coli based on the somatic (O), flagellar (H) and capsular polysaccharide antigens (K). The different antigens are unique for a particular serogroup. Uropathogenic E. coli is responsible for 90% of uncomplicated UTIs in otherwise healthy individuals. Various groups of O and H antigens have been associated with specific E. coli pathotypes. Ten out of 176 O serogroups are preferentially associated with Uropathogenic E. coli or UPEC. UPEC isolates, 536 and J96 both have been well studied, and they belong to phylogenetic group B2 and have serotypes, O6:H31 and O4:H5 respectively. The group of E. coli, often called UPEC, which are responsible for acute and chronic UTIs are distinct from the other E. coli that are commensal and are found in the colon of humans. These are represented by a few serogroups (O1, O2, O4, O6, O7 and O75). They often possess genes encoding many pathogenicity-associated factors including adhesins, siderophores or aerobactin, capsule and toxins implicated in UTI pathogenesis. PAIs as a distinct type of genetic element were first described for UPEC strain 536 (O6:K15:H31), which has become a model organism to study ExPEC pathogenesis ((Lloyd, AL., Rasko, DA., and Mobley, HLT., 2007). Reference List Beekmann, S. E., Diekema, D. J. and Doern, D. J., ( 2005). Determining the clinical significance of coagulase-negative staphylococci isolated from blood cultures. Infect. Control Hosp. Epidemiol. 26:559-566. Chapin, K., and M. Musgnug. (2003). Evaluation of three rapid methods for the direct identification of Staphylococcus aureus from positive blood cultures. J. Clin. Microbiol. 41:4324-4327. Johnson, JR., Owens, K., Gajewski, A., and Kuskowski, MA., (2005). Bacterial Characteristics in Relation to Clinical Source of Escherichia coli Isolates from Women with Acute Cystitis or Pyelonephritis and Uninfected Women. J. Clin. Microbiol.; 43: 6064 - 6072. Lloyd, AL., Rasko, DA., and Mobley, HLT., (2007). Defining Genomic Islands and Uropathogen-Specific Genes in Uropathogenic Escherichia coli. J. Bacteriol.; 189: 3532 - 3546. Ruimy, R. et al, (2008). Accuracy and Potential Usefulness of Triplex Real-Time PCR for Improving Antibiotic Treatment of Patients with Blood Cultures Showing Clustered Gram-Positive Cocci on Direct Smears. J. Clin. Microbiol.; 46: 2045 - 2051. Read More