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In this thesis, colonization, carriage and transmission of Staphylococcus aureus (S. aureus) were studied. A novel experimental decolonization and carriage model in rhesus macaques (Macaca mulatta) was set up. We showed that we could decolonize the nose of rhesus macaques that carried S. aureus, for a period of 10 weeks or more and we were able to inoculate the noses of the rhesus macaques with human S. aureus strain 8325–4. Another study with the human inoculation model was performed and methicillin-susceptible S. aureus (MSSA) strain 5062 of bovine origin (ST398, CC398) was found capable of surviving in the human nose for at least 21 days and it successfully outcompeted a human strain 1036 (ST931, CC8). We investigated, using whole-genome sequencing (WGS), whether nosocomial acquisition of S. aureus via healthcare workers (HCWs) occurred in neonates admitted to a neonatal intensive care unit (NICU). We also studied whether transmission and the presence of specific virulence genes support the occurrence of neonatal bloodstream infections. With our data, we can confirm that HCWs might be an important reservoir and link in the transmission of MSSA to neonates and this could partly explain the occurrence of neonatal S. aureus bacteremia.

In this thesis, colonization, carriage and transmission of Staphylococcus aureus (S. aureus) were studied. A novel experimental decolonization and carriage model in rhesus macaques (Macaca mulatta) was set up. We showed that we could decolonize the nose of rhesus macaques that carried S. aureus, for a period of 10 weeks or more and we were able to inoculate the noses of the rhesus macaques with human S. aureus strain 8325–4. Another study with the human inoculation model was performed and methicillin-susceptible S. aureus (MSSA) strain 5062 of bovine origin (ST398, CC398) was found capable of surviving in the human nose for at least 21 days and it successfully outcompeted a human strain 1036 (ST931, CC8). We investigated, using whole-genome sequencing (WGS), whether nosocomial acquisition of S. aureus via healthcare workers (HCWs) occurred in neonates admitted to a neonatal intensive care unit (NICU). We also studied whether transmission and the presence of specific virulence genes support the occurrence of neonatal bloodstream infections. With our data, we can confirm that HCWs might be an important reservoir and link in the transmission of MSSA to neonates and this could partly explain the occurrence of neonatal S. aureus bacteremia.

BACKGROUND: Neonatal Staphylococcus aureus (S. aureus) bacteremia is an important cause of morbidity and mortality. In this study, we examined whether methicillin-susceptible S. aureus (MSSA) transmission and genetic makeup contribute to the occurrence of neonatal S. aureus bacteremia. METHODS: A retrospective, single-centre study was performed. All patients were included who suffered from S. aureus bacteremia in the neonatal intensive care unit (NICU), Erasmus MC-Sophia, Rotterdam, the Netherlands, between January 2011 and November 2017. Whole-genome sequencing (WGS) was used to characterize the S. aureus isolates, as was also done in comparison to reference genomes. Transmission was considered likely in case of genetically indistinguishable S. aureus isolates. RESULTS: Excluding coagulase-negative staphylococci (CoNS), S. aureus was the most common cause of neonatal bacteremia. Twelve percent (n = 112) of all 926 positive blood cultures from neonates grew S. aureus. Based on core genome multilocus sequence typing (cgMLST), 12 clusters of genetically indistinguishable MSSA isolates were found, containing 33 isolates in total (2-4 isolates per cluster). In seven of these clusters, at least two of the identified MSSA isolates were collected within a time period of one month. Six virulence genes were present in 98-100% of all MSSA isolates. In comparison to S. aureus reference genomes, toxin genes encoding staphylococcal enterotoxin A (sea) and toxic shock syndrome toxin 1 (tsst-1) were present more often in the genomes of bacteremia isolates. CONCLUSION: Transmission of MSSA is a contributing factor to the occurrence of S. aureus bacteremia in neonates. Sea and tsst-1 might play a role in neonatal S. aureus bacteremia.

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE.

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE.

Our human model of nasal colonization and eradication of S. aureus is limited by safety issues. As rhesus macaques are closely related to humans and natural hosts for S. aureus, we developed an experimental decolonization and inoculation protocol in these animals. Animals were screened for nasal carriage of S. aureus and 20 carriers were selected. Decolonization was attempted using nasal mupirocin (10 animals) or mupirocin plus trimethoprim/ sulfadiazine intramuscularly (10 animals) both once daily for 5 days, and checked by followup cultures for 10 weeks. Intranasal inoculation was performed with S. aureus strain 8325–4 in culture-negative animals. 11/20 animals, of which 5 received mupirocin and 6 the combination treatment, became culture-negative for S. aureus for 10 weeks and these 11 animals were subsequently inoculated. Swabs were taken once a week for 5 weeks to test for the presence of the inoculated strain. In 3 animals, strain 8325–4 was cultured from the nose 1 week after inoculation, indicating short-term survival of this strain only, a finding similar to that previously found in our human model. These data demonstrate that rhesus macaques may constitute a relevant animal model to perform S. aureus eradication and inoculation studies with relatively limited invasive handling of the animals.

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer. We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2-21 days) than the bovine strain 5062 (median 21 days; range 7-21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.