Medical Genetics

STUDENT NAME

November 25, 2020

CLASS NAME

Introduction

Staphylococcus aureus is a part of normal skin flora but can cause invasive infections of the skin, soft tissue, and blood stream. It is the leading cause of health care associated infections (Coll, 2017). Healthcare workers have an increased likelihood of being asymptomatic carriers of disease (Alkharsah, 2018). With development of resistant strains, like MRSA, this can confer a major public health threat if an outbreak arises, as treatment options become more limited and more expensive.

This lab examines the case of an infant who was transferred to the neonatal intensive care unit with fever, refusing to feed, increased rate of breathing, increased heart rate, and swelling and redness of the umbilical stump. An infectious work-up was begun, as a systemic infection was suspected. Blood cultures were obtained and grew a Gram positive coccus. Three healthcare workers and two other infants later tested positive for MRSA carriage. MRSA isolates were obtained for whole genome sequencing to determine the spread among isolates and whether there had been an outbreak in the NICU. This lab worked to determine the possibility of cross-infection in the unit through culture analysis and genomic sequencing.

Methods

Blood cultures and umbilical cord swabs were obtained and plated on agar. A series of follow-up tests were performed, including motility, anaerobic growth, catalase, glucose, oxidative-fermentation, and agglutination. Antimicrobial sensitivity testing and phage typing was performed. Finally, genomic sequencing of all collected samples was performed.

Results

The agar plate Gram stain testing demonstrated Gram positive cocci growing from both the umbilical cord stump sample and the blood sample. A series of biochemical tests were then performed to attempt to speciate the bacteria. A motility test was then performed on the bacteria to assess for the presence of flagella. This testing demonstrated non-motile bacteria. The bacterium was found to grow both anaerobically and aerobically, defining it as a facultative anaerobe. The catalase enzyme test demonstrated oxygen bubbling, suggesting the bacteria to be catalase-positive and capable of breaking hydrogen peroxide down to water and oxygen. The glucose (acid) test showed no color change, indicating a lack of acid production and therefore a neutral pH. The oxidative-fermentation, or carbohydrate, test was performed to differentiate between the oxidative and fermentative breakdown of carbohydrates. This test demonstrated yellow color change for both open and closed tubes, thereby demonstrating the bacteria’s ability to ferment. Finally, agglutination testing was performed to test for coagulase presence; these results were positive for coagulase presence. Coagulase positivity indicates S. aureus as the pathogen.

Antimicrobial sensitivity testing was performed using MH agar and disc susceptibility. Clear zones of inhibition were identified and measured. In both umbilical and blood samples, the inhibition zone diameters were less than the clinical breakpoint for cefoxitin, norfloxacin, tobramycin, erythromycin, and clindamycin. Only fusidic acid produced diameters above the clinical breakpoint. These diameters are listed in Table 1.

Minimum inhibitory concentration (MIC) values were then determined for two antibiotics: oxacillin and vancomycin. An oxacillin MIC of >2 mg/L is suggestive of MRSA. The MIC for oxacillin was found to be greater than 256 mg/L and 0.5 for vancomycin.

Phase typing of the MRSA isolates from the NICU was performed in order to determine the possibility of cross-infection with concern for an outbreak. Phage types 7a, 7b, and 17 were isolated from the patient. MRSA phage typing results from the three healthcare workers and two other infected babies all showed phage types 7a, 7b, and 17, as well.

Genomic testing showed that the umbilical and blood sample isolates were both methicillin resistant S. Aureus. The samples were the same in BLAST sequencing and the mecA gene was identified in the sample, indicating methicillin resistance. Antimicrobial resistance testing was then performed through in silico analysis. The isolated blood sample demonstrated antibiotic resistance to all penicillin derived compounds. Finally, a phylogenic tree was constructed using the single nucleotide polymorphisms demonstrated amongst the strains, as detailed in Figure 1. This showed close relation among all infected healthcare workers, out patient, and the other two babies.

Figure 1: Phylogenic tree of all isolated samples.

Discussion

This child demonstrated infection in both the umbilical stump swab and the blood sample. The likely mode of transmission was from the infected stump seeding the blood, initiating a blood-borne infection.

The presence of a gram-positive coccus that is non-motile, catalase-positive, acid-positive, fermentative, and a facultative anaerobe aligns with findings of the Staphylococcus species. Agglutination testing then demonstrated coagulase positivity, suggesting infection with Staphylococcus aureus, specifically.

The results of the antimicrobial sensitivity testing showed a zone of inhibition above the clinical breakpoint only for fusidic acid. This suggests susceptibility to this antimicrobial and resistance to the other antibiotics: cefoxitin, norfloxacin, tobramycin, erythromycin, and clindamycin. An MIC >2 mg/L to oxacillin is suggestive of MRSA. The MIC found in this case study was 256 to oxacillin, suggesting MRSA infection.

Given the results of the MIC testing, vancomycin is the most appropriate drug to begin this patient on for treatment. The bacteria was found to be resistant to all of the tested antibiotics except vancomycin. This is also the initial treatment of choice for MRSA bacteremia, or blood-borne MRSA infection, as is seen in our patient (Choo, 2016).

The MRSA samples of the three healthcare workers and two infants showed identical phage typing to that of the patient: 7a, 7b, 17. Bacteriophages are viruses that infect and kill bacteria with a very strict host range. Isolates with the same phages are likely linked and part of an outbreak. The results of this test suggests an outbreak present in the NICU with the same MRSA strain. Because the patient was the first to show symptoms, the healthcare workers may have contracted the MRSA infection from the patient and later transferred the infection to the other two babies. MRSA grows on the skin and in the nares, so it is highly contagious. A second possibility remains that the healthcare workers are carriers, as the incidence of MRSA carriers is increased in hospital workers (Alkharsah, 2018). Carriers are asymptomatic, like those in this case, but still capable of transmitting disease that may present as infectious symptoms in others, like our patient.

An emergency admission required real-time PCR to screen a new admission for MRSA. This is a much faster means, and therefore preferred means, for screening than cultures. Cultures are preferable in a known or suspected site of infection. If this child screened positive and was asymptomatic, no treatment would be needed, as this would likely indicate carrier status. Genomic sequencing could be performed to check for an outbreak if that concern arises.

Genomic testing is performed in suspected outbreaks in isolated hospital setting, such as the case in this scenario (Coll, 2017). The mecA gene was found, indicating low affinity for penicillin binding (Chambers, 2009). This confers the “methicillin resistant” property of MRSA. The in silico analysis demonstrated genotypic resistance to the penicillin-derived compounds. This is congruent with the phenotypic results demonstrated earlier and also what is expected of MRSA. The mecA gene defines MRSA and provides the bacteria with resistance to penicillin agents.

A number of virulence factors have been identified in MRSA, including Panton-Valentine leukocidin (PVL), alpha-toxin, alpha-helical peptides (PSMs), various genetic elements, biofilms, and arginine catabolic mobile element (ACME). PVL is an exotoxin associated with abscess formation that is carried by nearly every MRSA strain. Its exact role is controversial. The alpha-toxin is a pore-forming leukocyte toxin that is well described in S. aureus strains. It alters platelet morphology and lyses white blood cells. PSMs recruit, activate, and lyse neutrophils in high concentrations and initiate pro inflammatory responses. Chromosomal genetic elements, like the mecA gene discussed previously, are also important virulence factors. ACME plays an important role in the pathogenesis, growth, and transmission of MRSA. Superantigens can cause serious toxicity, like toxic shock syndrome. These initiate a cytokine storm and contribute to biological fitness of MRSA. MRSA also has an ability to create biofilms. This can complicate infections, especially those of foreign materials, like prosthetic joints, and make them especially difficult to eradicate (Watkins, 2012). In this patient, particular concerns would be the chromosomal genetic mecA gene that conferred methicillin resistance. PVL, alpha-toxins, and ACME may also be implicated.

The branching seen in the phylogenic tree suggests that a healthcare worker initiated the outbreak that ultimately led to the case of neonatal sepsis. He/she is a carrier for the disease and likely spread the infection unknowingly amongst other workers and patients. MRSA is commonly associated with health care or hospital associated transmission. A study from 2010 assessed MRSA infections from 2005-2008 and showed that of 21,503 invasive MRSA infections, 17,508 were health care associated (Kalen, 2010). This is a major public health concern that must be addressed within hospitals and health care institutions through proper hand hygiene, careful cleaning of equipment and hospital rooms, and contact precautions used in patients with known MRSA infections. The difficulty in the presented case is 1) carrier status and 2) unknown pathogens at early infection.

Between one-fifth to one-third of the population are Staph carriers and about 2-3% of people carry methicillin resistant strains. This is increased in health care workers who have increased exposure to disease. Carriers are asymptomatic but still capable of spreading infection (Alkharsah, 2018). Most people do not know they are carriers, however, and therefore may not be taking precautions to stop disease spread. While rapid PCRs can be performed to identify carriers, carriers are not treated for the infection. Therefore, the best prevention that can be taken by hospitals and health care workers is through proper hand hygiene, thorough cleaning of hospital rooms and equipment, and proper contact precautions.

Another difficulty in this case and all infectious outbreaks is a delay in symptom presentation. While others contracted the MRSA infection from the infected infant, the infant was likely colonized with MRSA before she showed any symptoms. Healthcare workers are more inclined to take precautions, like strict contact regulations, when a known infection is present.

A MRSA outbreak is underway in the NICU but its course can be stopped and reversed through improved hygiene, improved contact precautions, and the initiation of vancomycin to symptomatic individuals.

REFERENCES

Alkharsah, K.R., Rehman, S., Alkhamis, F. et al. Comparative and molecular analysis of MRSA isolates from infection sites and carrier colonization sites. Ann Clin Microbiol Antimicrob 17, 7 (2018). https://doi.org/10.1186/s12941-018-0260-2

Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. 2009;7(9):629–41.

Choo EJ, Chambers HF. Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia. Infect Chemother. 2016;48(4):267-273. doi:10.3947/ic.2016.48.4.267

Coll, F., Harrison, E.M., Toleman, M.S., Reuter, S., Raven, K.E., Blane, B., Palmer, B., Kappeler, A.R.M., Brown, N.M., Török, M.E., Parkhill, J. and Peacock, S.J. (2017). Longitudinal genomic surveillance of MRSA in the UK reveals transmission patterns in hospitals and the community. Science Translational Medicine, 9(413), p.eaak9745.

Kallen AJ, Mu Y, Bulens S, et al. Health Care–Associated Invasive MRSA Infections, 2005-2008. JAMA. 2010;304(6):641–647. doi:10.1001/jama.2010.1115

Watkins RR, David MZ, Salata RA. Current concepts on the virulence mechanisms of meticillin-resistant Staphylococcus aureus. J Med Microbiol. 2012;61(Pt 9):1179-1193. doi:10.1099/jmm.0.043513-0