Epidemiological background:

The outbreak occurred in the first week of February 2023, during which time 6 patients developed pneumonia and a further 8 patients and 2 staff members developed milder, flu-like symptoms. Once a diagnosis has been achieved, all patients were prescribed erythromycin. Four of the 6 pneumonia patients made a rapid recovery, but two patients required life-support in the intensive care unit (ICU). One of these patients was immediately prescribed levofloxacin and made a rapid recovery, but the condition of the second patient, who was treated with azithromycin, did not improve until his therapy was switched to levofloxacin.

A PHE outbreak investigation team collected samples from various water sources in the hospital in order to try and identify where patients acquired their infections from.

Patient Diagnostics:

Infection was diagnosed by isolation ofLegionella pneumophila(L. pneumophila) from patient sputum. This was achieved by direct inoculation of sputum onto on buffered charcoal yeast extract agar, then incubation of plates for 48 hours. Isolates were initially identified asL. pneumophilaon the basis of colonial morphology and Gram staining, then confirmed using aL. pneumophila-specific latex agglutination test.L. pneumophilaisolates were obtained from all six of the pneumonia patients but none of the patients or staff with mild flu-like symptoms. No evidence of SARS-CoV2, influenza or any other infection apart fromL. pneumophilacould be found in any of the patients and staff at the hospital during the outbreak.

Environmental sampling:

12 water samples were collected from the hospital. Each sample was processed according to British Standards Institute BS 7592. Isolates ofL. pneumophilawere obtained from 6 samples collected from different water sources in the hospital.

Fingerprinting of isolates:

Clinical and environmentalL. pneumophilaisolates were serotyped and subjected to multi-locus sequence typing (MLST). The results obtained are provided in the Table below:

Isolate	Serotype	MLST type
Patient 1	1	21
Patient 2	1	21
Patient 3	1	21
Patient 4	1	21
Patient 5	1	21
Patient 6	1	21
Birthing pool	1	29
Shower 1 (for Patients)	1	13
Shower 2 (for Patients)	1	21
Shower 3 (for Staff)	1	21
Air conditioning system	1	21
Water fountain	1	19

Antibiotic susceptibility testing:

The susceptibilities of all clinicalL. pneumophilaisolates were tested against a range of antibiotics. Minimum inhibitory concentrations (MICs) are provided in the Table below:

Isolate	Antibiotic
erythromycin	rifampicin	ciprofloxacin	doxycycline
Patient 1	0.25	0.016	1	0.5
Patient 2	0.5	0.016	1	0.5
Patient 3	0.25	0.032	1	1
Patient 4	1	0.016	2	0.5
Patient 5	1	0.008	2	1
Patient 6	0.5	0.008	2	1

Refer to EUCAST antimicrobial susceptibility testing ofLegionella pneumophilaguidance (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Legionella_guidance_document_20171208.pdf) for help in interpreting these results.

Whole genome sequencing:

Whole genome sequencing was performed on 2 clinical isolates and 3 environmental isolates and comparison of single nucleotide polymorphisms (SNPs) in 1809 genes within theL. pneumophilacore genome was performed. A dendrogram of relatedness based on this analysis was generated:

Whole genome sequencing also demonstrated the presence of thelpeABgene in the genome ofL. pneumophiliaisolated from patient 5 but this gene was absent from genome ofL. pneumophiliaisolated from patient 1.

Requirements of review and questions to answer in full detail:

1. Assess the performance of the diagnostic methods used in terms ofpatient management. This assessment should also consider the availability and performance of alternative assays that may have improved patient management. This assessment should consider both culture-based and non-culture based diagnostic methods. Do not forget to consider patients and staff with flu-like symptoms in your assessment.
2. Among the 6 water sources from whichL. pneumophilawas recovered, which do you think is the most likely environmental origin of the outbreak? Explainin detailwhy you reached this conclusion and why other potential water sources were discounted on the basis of epidemiological and fingerprinting results. This explanation should (i) consider the likelihood of each source from a practical perspective, (ii) summarise what each of serotyping, MLST and whole genome SNP analysis actually is and thus what the differences between them are, and (iii) refer to the contribution of each to determining the fingerprints ofL. pneumophilain this scenario.
3. Briefly summarise how water samples were tested for the presence of legionellae. Include detail of sample were collected and processed in the laboratory. Explain why using BS7592 is important. The hospital has a legal obligation to control legionella on its premises; the requirements for doing so are outlined in the Health and Safety Executive's INDG458 publication (and elsewhere). Briefly summarise the duties the hospital must perform to fulfil these requirements.
4. Summarise how the success and failure of the antibiotics used to treat patients during the outbreak can be explained on the basis of the antibiotic susceptibility test results and relevant data obtained from whole gene sequencing. This discussion requires you to access and read current guidelines for the treatment of legionellosis in the UK and research studies intoL. pneumophilaAMR. Do these results align with those in other studies that have explored antibiotic susceptibility/resistance ofL.pneumophila?Please describe in detail all the scenario and answer the relevant questions to it and also reference as you go along. Many thanks!!