Report A, Case study 1:

Report A, Case study 1:

Abstract:

A serious situation within the NICU occurred when a 6-year-old infant presented symptoms of fever, rapid breathing of (>60 breaths/ minutes), a rapid heart rate of (>200 beats/minutes) and signs of inflammation in the umbilical cord stump. There seems to be a systemic infection, confirmed after taking blood samples and incubating for 16 hours. The results were flagged positively as the blood culture presented a gram-positive coccus. There is a possibility of an outbreak primarily due to three healthcare workers and two other infants that have tested positive for MRSA, fearing a high chance of cross-infection within the NICU and a high possibility of MRSA as the source of illness.

In the investigation, we aim to diagnose and initiate appropriate treatments by determining the pathogen causing the systemic infection. Identifying the source of the outbreak by establishing the epidemiological relationship between MRSA isolates to determine if there are outbreaks and to specify where it is coming from in the NICU.

Differential diagnosis could include bacterial sepsis, in view of the infants fever, rapid breaths and the positive blood culture. Following the treatments could involve broad-spectrum antibiotics whilst waiting for the culture results as the samples collected such as the blood sample, for the cultures and whole genome sequencing (WGS) and MRSA isolate can aid in the confirmation of diagnosis and identify the specific cause.

When confirming the overall diagnosis, we need to analyse the blood cultures further and perform further tests, such as a sensitivity test to select the appropriate antibiotic. To treat the infant, targeted antibiotics could be involved depending on the pathogen, as well as monitoring closely with supportive care put in place. The whole genome sequencing of MRSA isolates are essential to confirm the outbreak, comparing the sequences generated specifying whether the strains are related and will confirm the epidemiological link. Nonetheless, the prognosis entirely depends on early diagnosis with suitable treatment put into place alongside control measures in the NICU, further preventing spread of infection ((Odabasi and Bulbul, 2020) and (MISRA, SINGH and MISRA, 1997)).

Word count: 329.

Question (1): How did macroscopic examination of agar plates contribute to the identification of the pathogen isolated from the infant?

Answer:

Agar plates are petri dishes that grow bacteria and fungi in microbiology laboratories. Examining agar plates macroscopically aids in identifying pathogens and makes it essential to recognise the activities of pathogens by being aware of the morphology, growth patterns and metabolic activity. Methicillin-resistant Staphylococcus aureus (MRSA) was screened using a standard culture and plated on the chromogenic media. A suspected pathogen of S. aureus colonies was present as the media displayed a denim blue tint, indicating a gram-positive bacterium, as is shown in both Figure 1a and 1b as they are formed together in clusters (Peterson et al., 2009).

Blood agars are typically used to test organisms abilities to produce haemolysin, enzymes that are able to lyse red blood cells. S. aureus usually produces at least three haemolytic activities: , , and . To ensure a pure isolate of the pathogen, the morphology of colonies on the blood agar presented a -haemolysis (gamma haemolysis) (Ejiofor et al., 2018). As shown in the figure below in both 1a and 1b, the blood agar is presented to be none haemolytic; therefore, it is y-haemolysis (Wang et al., 2020).

Figure 1a: Umbilical cord sample Figure 1b: Blood sample

Figure legend 1a and 1b: presented in 1a and 1b are all three tests conducted, the chromogenic media, blood agar and nutrient agar. The spectra MRSA Chromogenic media was plated with methicillin-resistant staphylococcus aureus to identify it based on the colonies colour, which displayed denim blue. The blood agar presents a gamma haemolysis.

Question (2): Discuss the principle of the chromogenic medium used in the identification of the patients pathogen? And what are the advantages of using chromogenic media for bacterial identification?

Answer:

There are advantages of chromogenic media it can utilise synthetic chromogenic enzyme substrates so that they can specifically target pathogens based on enzymatic activity and depending on the ability to create specific enzymes or metabolites. The majority of the chromogenic media are both selective and differentiated, allowing them to adapt to the inhibition of nontarget organisms and allowing the target enzymes to grow as coloured colonies for better identifications. Based on the metabolism of chromogenic enzyme substrates. When only the targeted colonies generate a colour, it effectively reduces time spent on investigating nontargeted colonies compared to other culture media tests as it employs colourless enzymes that can be hydrolysed by specific microbes that result in invisible colour changes which produce products in growing colonies or those surrounding the colonies ((Xu et al., 2016 and (Perry, 2017)). This method effectively saves costs ranging from labour time and the use of regents as fewer biochemical tests are required to be carried out as it contributes to a much quicker confirmation of the pathogen (Perry, 2017).

Question (3): Describe the results of Gram staining. And briefly discuss the principle of Gram staining in the identification of the pathogen isolated from the patient.

Answer:

Gram staining is a crucial method used in microbiology and within this investigation. The gram staining involves using crystal violet or methylene blue as a primary colour. Organisms that retain the primary stain and appear blue-purple are gram-positive, whereas the organisms retaining a red-pink stain are gram-negative, either possessing a bacilli that are rod-shaped or cocci, which are spherical in shape. They are usually used to indicate whenever a bacterial infection is suspected, which would aid in identification. As presented in Figure 2, due to the result of the gram staining the bacteria is retaining the stain of blue-purple in which we can confirm its a positive cocci in a spherical shape in clusters, a usual indication of staphylococcus such as S. aureus. The basic principle of gram staining is the ability of the bacterial cells and their ability to retain the crystal violet dye during the solvent treatment. Gram-positive microorganisms have a higher peptidoglycan content compared to gram-negative organisms as they have a higher lipid content. Originally all bacteria are able to retain the crystal violet dye, but using solvents, lipids from gram-negative organisms are dissolved, thus losing the primary stain. A stark contrast to gram-positive organisms, as the solvent dehydrates the gram-positive cell walls as the closure of pores prevents any diffusion of violet-iodine complex therefore, the bacteria continues to be stained (Tripathi and Sapra, 2023).

Figure 2: the result presents a blue-purple/brown Gram-positive cocci in clusters, indicating the suspected presence of S. aureus.

Question (4): Use a table to summarise the results of biochemical tests used for the identification of the genus of bacterium isolated from the patient. And discuss in detail which laboratory test was used for the identification of bacterial species.

Answer:

To identify the bacterial species, the following tests were conducted as presented in Table 1. The specific tests used for identifying the bacterial species infecting the infant was the coagulase agglutination test. The test came back positive, which is an indication to staphylococcus aureus. S. aureus have strains that can secrete free plasma coagulase, which is considered to an important virulence factor for the bacteria. Tube coagulase tests take a long time up to 24 hours to generate a result, whereas the rapid S. aureus coagulase takes seconds to generate result to confirm diagnosis, serving its purpose for getting developed. The test has particles precipitate with a few S. aureus surface antigens which allow S. aureus isolates and CNS (coagulase-negative staphylococci) isolate to differentiate between them faster. When tests prove the presence of S. aureus, as the results show in Table 1, further tests are performed for methicillin resistance (Weist, 2006).

Table 1: biochemical test results

Tests Results

Gram stain Gram positive cocci

Growth in the air/ anaerobically Facultative anaerobe

Catalase Positive

Glucose (acids) Positive

Carbohydrates (F/O/-) Fermentative

Coagulase agglutination Positive

Figure 3: coagulase agglutination tube test control, positive results for both blood sample and umbilical samples.

Question (5): Use a table to present the results of antimicrobial susceptibility testing (AST) for both bacterial isolates detected in the patient. And discuss in detail how AST confirmed bacterial identification.

Answer:

Antimicrobial susceptibility testing (AST) would be able to specify the effective dosages needed and formulate a profile for practical therapy options to manage deadly bacterial infections. The minimum classified concentrations (MIC) of AST testing that are categorised using the guidelines set out by the clinical and laboratory standard institutions (CLSI) based on the pharmacokinetics-pharmacodynamics (PK-PD), which presents the properties and the mechanisms of resistance for the guideline cut of breakpoint (Khan, Siddiqui and Park, 2019). According to GUIGNARD, ENTENZA and MOREILLON (2005), MRSA resistance is commonly linked to the element SCCmec, essentially the gene which encodes for the penicillin-binding protein 2A (PBP2A), as it has a low affinity of B-lactam.

The tables

Table 2a: antimicrobial susceptibility tests (AST), blood culture

Antibiotic Clinical breakpoint Case study strain, blood culture Sensitive or resistant

Cefoxitin FOX 30g ug 24 19 Resistant

Norfloxacin (NOR)10ug 20 6 Resistant

Tobramycin (TOB) 10 ug 32 23 Resistant

Erythromycin E 15ug 21 6 Resistant

Clindamycin (DA) 2ug 40 30 Resistant

Fusidic acid (FD) 10ug24 28 Sensitive

Table 2b: antimicrobial susceptibility tests (AST), umbilical cord

Antibiotic Clinical breakpoint Case study strain, blood umbilical cord Sensitive or resistant

Cefoxitin FOX 30g ug 24 19 Resistant

Norfloxacin (NOR)10ug 20 6 Resistant

Tobramycin (TOB) 10 ug 32 23 Resistant

Erythromycin E 15ug 21 6 Resistant

Clindamycin (DA) 2ug 40 30 Resistant

Fusidic acid (FD) 10ug24 28 Sensitive

Table 3: Minimum inhibitory concentration (MIC)

Antibiotic MIC blood culture MIC umbilical

Oxacillin >256 >256

Vancomycin 0.5 0.5

Question (6): Present the results of phage typing and discuss their significance in detail.

Answer:

The phage typing result

Figure 4:

Question (7): Describe the results of real-time PCR and discuss why this test was carried out and mention its advantages and limitations.

Answer:

g

Figure 5:

Figure 6:

Question (8): Use a table to present the results of in silico analysis of antibiotic resistance of the blood culture strain isolated from the infant. And discuss in detail if there is an agreement between the results of genotypic analysis and phenotypic analysis of antibiotic resistance

Answer:

There is an agreement

Antibiotic (disc content) WGS-predicted phenotype Genotypic analysis Class

Cefoxin FOX 30ugResistant MecA (MecA_BX571856) Beta-lactam

Norfloxacin NOR 10ug

Sensitive No resistance gene Quinolone

Tobramycin TOB 10ug Resistant aadD (aadD_M19465) Aminoglycoside

Erythmoycin E 15ugResistant Erm(A)

(erm(A)_X03216) Macrolide

Clindamycin DA 2ug Resistant Erm(A)

(erm(A)_X03216) Linosamide

Fusadic acid FD Sensitive No resistance gene Steroid antibacterial

Question (9): What is the overall diagnosis of the case?. And what is the prognosis?. List the name of 3 virulence factors harboured by the genome of isolated pathogen from the patient. Discuss in detail the role of ONE of these virulence factors in bacterial pathogenicity.

Answer:

Question (10): Based on AST results, discuss in detail the recommended treatment for the case.

Answer:

Question (11): Provide a dot plot of the alignment between the complete genome of bacterial isolates from blood and umbilical cord. Discuss the significance of results in detail and if results can provide insights on how the infant might have acquired the infection?

Answer:

Figure 7:

Question (12): Present the alignment results of mecA gene (including nucleotide bases 1 to 480) against the genome of umbilical culture isolate?. Discuss the significance of results in detail.

Figure 8:

Question (13): According to in silico analysis results of antibiotic resistance, the isolated pathogen from the case showed no resistance against the following antibiotics; fusidic acid, ciprofloxacin and colistin can any of these antibiotics be used in the treatment? justify your answer.

Question (14): Provide a fully labelled phylogenetic tree showing the epidemiological relation among all MRSA isolates. Describe the results of phylogenetic analyses and discuss if there an outbreak of MRSA in the NICU.

Question (15): Mention what might happen in the neonatal intensive care unit to facilitate cross-infection. Discuss the control measures and what must be done to prevent cross-infection.

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