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Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals

NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001

Prepared by: Niall De Lappe Issue Date: 11.11.2019 of 14

Q-Pulse Document Control Authorised by: Martin Cormican

CURRENT VERSION AMENDMENTS Each Policy and Procedure has an individual record of amendments. The amendments

for the current version are listed below.

Amendment

Number/ Date

Version no.

Discarded

Version

no. Issued

Section Amendment

11/11.11.19 4

4.1 Contact

details

Remove [email protected]

Services

offered

Remove Listeria serotyping

WGS Amended reference to wgs starting dates

Antibiotic

resistance

Updated

Specimen

rejection

Repeat isolates from same site rejected if within

3 months of previous isolate

New

section

Bioinformatics software used

Change Control No: MIC032/19

mailto:[email protected]





National Salmonella, Shigella & Listeria Reference

Laboratory

Users Guide

Contact details

Head of Department: Prof. M.Cormican (091) 544146

Laboratory Tel: (091) 544628

Fax: (091) 542238

e-mail: [email protected]

[email protected]

Address

National Salmonella, Shigella & Listeria Reference Laboratory

Department of Medical Microbiology

University Hospital Galway

Galway

Role of NSSLRL and relationships with other Agencies

Fig.1 NSSLRL Organisational Chart

Primary clinical laboratories identify Salmonella, Shigella and Listeria to species

level and determine the isolates susceptibility to those antimicrobial agents

Clinical Labs

NSSLRL

Public Health

HPSC ECDC

CDC

FSAI

Veterinary Labs Food Labs







immediately relevant to patient treatment. This provides immediate information to the

clinician for treating an individual patient.

Isolates are referred to the NSSLRL for confirmation and for subtyping. The NSSLRL

confirms the result of the clinical laboratory.

The NSSLRL also adds a national public health dimension to the work of the clinical

laboratories by recognition and confirmation of links between individual cases of

infection, even where outbreaks are widely dispersed. This information is primarily

to guide public health intervention to recognise and control transmission of infection.

Trends of Salmonella infections in Ireland can also be more closely followed. Typing

of isolates from food and animal sources help in tracing the sources of infection.

The NSSLRL works in tandem with numerous agencies in protecting public health.

The laboratory which identifies the pathogen and any clinician involved in the care of

the patient is obliged to notify the case to Public Health (PH). The 9 PH departments

in the country regularly review surveillance data locally to determine if incidence is

increased or if trends are occurring in age groups, areas and concerns are discussed

with the NSSLRL. PH contacts the case to try and determine the source of infection

and give advice to prevent spread of infection to others. The above actions may be

carried out by medical staff from PH with the assistance of Environmental Health

officers. Stool specimens may subsequently be submitted to clinical laboratories to

determine if contacts of the cases are also infected (outbreak) and/ or food (and

occasionally water) samples may be sent to a food and water laboratory to try to

determine a source of infection. If a relevant pathogen is isolated in those laboratories

the isolates will be sent to the NSSLRL for comparison with the isolate from the first

case or outbreak. As part of the investigation a questionnaire is administered to

investigate possible risk-factors (contact with animals, occupation, etc), travel history

and recent food history. When more than one case is detected cross-comparison of

questionnaires may allow a putative source to be identified through statistical

analysis.

The Health Protection Surveillance Centre (HPSC) looks at national trends. The two

tier surveillance ensures local trends are picked up by PH and that national trends can

be detected by HPSC. When major outbreaks occur an outbreak control team (OCT)





may be set up at the discretion of the Director of Public Health (regional level) or by

the Health Protection Surveillance Centre (National Level). Staff of NSSLRL are

consulted regarding the need for an OCT and the consultant microbiologist from

NSSLRL (and sometimes other staff of NSSLRL) are involved as members of the

OCT to guide the interpretation of the microbiology results. Other agencies involved

include HPSC, PH and Food Safety Authority of Ireland (FSAI).

The NSSLRL and HPSC liase with the European Centre for Disease Control (ECDC)

to help identify and control international dimensions to outbreaks. This involves

sharing data, countries issuing outbreak alerts and monthly outbreak summaries.

Services offered

The NSSLRL only performs analysis on isolates received on nutrient agar bijoux. The

NSSLRL does not test primary samples, e.g. faeces, blood or food, therefore results

are qualitative and not quantitative.

The NSSLRL types isolates from food and animal sources as well as those of human

origin. The primary role of the NSSLRL is to protect human health. Clients should be

aware that if animal or food isolates are similar to those from sporadic or outbreak-

associated human isolates this information will be shared with relevant bodies, e.g.

Food Safety Authority of Ireland (FSAI), Health Protection Surveillance Centre

(HPSC) and European Centre for Disease Control (ECDC) when it is necessary to do

so to protect public health. In general users will be informed in advance of sharing

the source laboratory information with these agencies. These bodies may then require

further information from the client laboratories.

If users wish to use data from the NSSLRL in publications they must contact the

laboratory director at [email protected] or in his absence one of the scientific

staff.

Salmonella - Analysis of Whole Genome Sequences (non-accredited)

Shigella - Analysis of Whole Genome Sequences (non-accredited)

Listeria - Analysis of Whole Genome Sequences (non-accredited)






Ninety-five percent of samples will be reported within 28 days and specimens that are

identified by telephone call as urgent will be prioritized.

Whole Genome Sequencing

The speed and accuracy of sequencing has increased and the cost has decreased

dramatically in recent years. It is now possible to use sequencing of bacterial

pathogens in outbreak investigations. Analysis of whole genome sequences (WGS) is

rapidly replacing conventional phenotypic. Serotyping and antimicrobial

susceptibility testing was discontinued on Salmonella and Shigella isolates received in

the NSSLRL after the 01/10/18 as analysis of WGS replaced these tests. Listeria

serotyping was discontinued on Listeria monocytogenes isolates from beginning of

2019.

Our primary interest when analysing sequences is to determine relatedness between

bacterial isolates in a timely manner.

The most widely used methods are;

1) Whole genome multi locus sequence typing (wgMLST) or a variation such as

core genome (cg)MLST.

2) Single nucleotide polymorphisms (SNP) analysis.

wgMLST

MLST was first used in 1998 to type Neisseria meningitidis and has since been used

to type a huge number of pathogens. As initially developed MLST involved extracting

DNA from bacterial isolates, performing separate PCR reactions on a number of

internal fragments (450-500 base pairs) of housekeeping genes, purifying the PCR

products and sequencing the products using Sanger sequencing. The resultant

sequences are then analysed to determine the alleles at each locus. Each time a novel

allele is detected it is assigned a new number. The numbering system is sequential so

the distance between numbers does not correlate with degree of relatedness.

Differences in allele sequences can arise from point mutations, insertions or deletions

(Indels), recombination events or a combination of the above. A unique combination

of alleles at each locus, an “allelic profile” specifies the sequence type (ST). The

MLST allele sequences and allelic profiles are stored in various curated databases





worldwide and these are collected by the PubMLST site and made easily accessible

[http://pubmlst.org].

Fig. 2 Diagrammatic representation of Multi Locus Sequence Typing (MLST) of

S.Enteritidis.

Fig. 3 Variations in allele sequences arising by single point mutations (strain 2, locus

A &B), insertions and/or deletions or indels (strain 3 locus A &B) and inversions

(strain 4, locus A).

Most Salmonella serotypes are “monophyletic” this means that they consist of

variants of a common ancestral sequence type, e.g. S.Enteritidis = ST11

(5,2,3,7,6,6,11 ), S.Typhimurium = ST19 (10,7,12,9,5,9,2), S.4,[5],12:i:- = ST34

(10,19,12,9,5,9,2), while other serotypes, e.g. S.Newport, have multiple lineages

(polyphyletic) and consist of numerous, often unrelated, sequence types, e.g. ST118

(16,42,39, 2,43,45,36) and ST166 (5,14,6,12,5,14,58).

‘Locus’ (gene) Strain 1 Strain 2 Strain3 Strain 4 A ACTAGAGGGAA ACTAGAGGCAA ACT-GAGGGTAA ACGGGAGATAA

allele 1 allele 2 allele 3 allele 4

B TAGCCAGGGTC TAGCAAGGGTC TAGC---GGTC TAGGCAGGGTC

allele 1 allele 2 allele 3 allele 1

C, D, E, etc…. alleles 5,2,8… alleles 1,4,7… alleles 1,3,9… alleles 6,2,9…

5

2

3

7

6

6

11

ST11

http://pubmlst.org/





With the advent of WGS an isolates seven gene allele profile and sequence type can

be deduced from the genome sequence without having to do the separate PCRs. Also

instead of analysing just 7 housekeeping genes the number of genes examined can be

greatly increased to look at entire core genomes (genes present in the genomes of the

vast majority of that pathogen) cgMLST or whole genomes wgMLST incorporating

both core and accessory genomes. This obviously greatly increases the discriminatory

power of MLST from the conventional 7 gene MLST schemes.

The wgMLST schemes need constant curation for QC and assigning new allele

numbers. A major advantage is that results are easily comparable when laboratories

use the same schemes.

SNP analysis

wgMLST only takes account of coding sequences. SNP analysis takes account of

mutations throughout the genome. Short reads from isolates would always be

compared against closely related reference genomes, e.g. S.Enteritidis against a

S.Enteritidis reference, S.sonnei against a S.sonnei reference, etc. This method is

harder to standardise as results depend on the reference strain used.

Fig. 4 Dendrogram of hqSNP analysis of Shigella sonnei using BioNumerics

software.





Antbiotic resistance

Fig. 5 Analysis of resistance genes of Shigella isolates in BioNumerics.

Reports on isolates of Salmonella, Shigella and Listeria monocytogenes will include

sequence type (ST), predicted serotype (based on ST) and relevant resistance genes,

i.e. genes that encode resistances to the antibiotics currently tested for in the

NSSLRL, i.e. A (ampicillin), C (chloramphenicol), Su (sulphamethaxazole), T

(tetracycline), Tm (trimethoprim), Na (nalidixic acid), Cp (ciprofloxacin), Caz

(ceftazadime), Gm (gentamicin), Ctx (cefotaxime), Azt (azithromycin), Tig

(tigecycline), Mer (meropenem), Col (Colistin).

In most cases Shigella isolates that are resistant to azithromycin have both erm(B) and

mphA genes while Salmonella with either gene tend to be resistant.

Most high-level ciprofloxacin resistance in Salmonella and Shigella is accounted for

by gyrA and parC mutations. BioNumerics can detect these mutations in Salmonella

but external software, ResFinder, is used to look for these mutations in Shigella

isolates. If for clinical reasons a laboratory wishes us to check ciprofloxacin MICs

please contact us and we can perform broth microdilution on the relevant isolate.





Fig. 6 Minimum spanning tree (MST) of Salmonella Newport core genome MLST

from isolates from humans in the NSSLRL from 2015-18 coloured by designated

cluster.

S.Newport (6,8:e,h:2) is a polyphyletic serotype, i.e. isolates with this antigenic

structure have originated at different times so all did not evolve from a single

ancestor. The numbers on the branches indicates the number of allele differences

between isolates. Epidemiologically related isolates should have the same or closely

related cg and/or wgMLST profiles. Cluster C/17 isolates were fully susceptible to all

antimicrobials tested. Many of the cases were young adults and had a history of travel

to a tourist destination island. In contrast the isolates in cluster D/17 had resistance to

quinolones and were mainly associated with an older cohort in the East of the country.

[The code C/17 is NSSLRL designation for 3rd

such cluster identified in the year

2017]

1

13

60

67

24

28

13

2346

897

1493

107

2409

7

326

354

C/17

D/17





WGS data is continuously analysed for similarities between isolates and if any

evidence of outbreaks or linked cases are detected then the relevant public health

authorities will be informed.

Bioinformatics software used

Bioinformatics analysis in the NSSLRL is performed primarily with BioNumerics

software (Applied Maths) although other programs are used occasionally.

BioNumerics software 7.6.3

E.coli functional genotyping plug-in version1.2

Salmonella functional genotyping plug-in version 0.1

Listeria functional genotyping plug-in version 1.0

SeqSero

CGE software http://www.genomicepidemiology.org/

KmerFinder

ResFinder

PubMLST https://pubmlst.org/

Databases

Species identification by ribosomal MLST

EnteroBase http://enterobase.warwick.ac.uk/

Opening times

Mon – Fri 9:00am- 5:00pm (lunch from 1:00 – 2:00pm)

Out of hours work

This is performed at the discretion of the Consultant Microbiologist or the laboratory

scientific staff on urgent samples, e.g. during an outbreak investigation.

Laboratory Contact Out of Hours: (091) 544411

Specimen Rejection Policy

Specimens sent to the NSSLRL will be rejected if:

- isolates are sent on agar plates, plastic universals or large bijoux





- specimens contain a mixed bacterial culture

- specimen slope is broken

- specimen form and/or slope are unlabeled, mismatched or incomplete

- transportation of samples to the NSSLRL is not followed correctly, i.e.

slopes not enclosed in a crushproof container

external packaging not labeled correctly

- if for any other reason the specimen is not in a safe condition for processing

and/or can not be clearly identified.

- Duplicate isolates from patients from the same site (e.g. faeces) received within

three months of the initial isolate, even if they are from different hospitals. When

this occurs the sequencing results of the initial test will be included in the report

along with a note stating “An isolate from this patient was recently received from

another hospital. WGS was not performed on this isolate. The sequencing results

in this report are from the previous isolate”

- If a repeat isolate from the same site is received from the same hospital this will

be rejected with a note stating “An isolate from this patient was recently

sequenced. Please see report .....”.

- If isolates are received from multiple sites from the same patient WGS will be

performed on all isolates.

- Laboratories should send only one nutrient agar slope per patient site. If a

laboratory sends two slopes only one will be processed. The second slope will be

held and processed only if there is a problem with the first slope, e.g. no growth

or growth of a non-Salmonella/Shigella/Listeria isolate.

Minimum requirements for completion of NSSLRL request forms

NSSLRL request forms must be completed in legible handwriting or typing and all

details must be entered.

- Human isolates

The patient name and referring lab number and/or date of birth must be on

both the form and side of slope.





If there is reason to suspect the isolate could be a Salmonella Typhi or

Salmonella Paratyphi, e.g. patient from an endemic country, this must be

noted on the form.

If the isolate is suspected to be part of an outbreak this must be noted on the

form.

- Non-human isolates

The referring lab number and isolate source must be on both the form and side

of slope

Isolate source must be clearly stated, e.g. pork, chicken, beef, otherwise a

report will not be issued.

Transportation of samples

Slopes must be packaged and transported according to ADR (Carriage of Dangerous

Goods by Road) regulations.

Grow isolate overnight on a small nutrient agar slope and remove any fluid

accumulating at the bottom of the slope using a sterile Pasteur pipette.

Place slope(s) into an inner crushproof hard plastic container and place this

into a cardboard box or envelope.

Label with an emergency contact number for the sender and an Infectious

substance label.

Label box with UN3373 and add sticker with “BIOLOGICAL SUBSTANCE,

CATEGORY B”.

This can be then sent to the Reference laboratory via a courier company, e.g.

Hays DX (01-8421088), Capital Freight (01-8852064), Biomnis (1800-

252967) or Nightline couriers (091-795100), MedLab Pathology (01-2933690)

A number of slopes may be sent in each crush-proof container but each slope

must be individually wrapped in an adsorbent material to prevent breakage

during transit.





Retention times

Additional examinations may be requested during specimen storage time by

telephoning the NSSLRL. Agar slopes (apart from CL3 isolates) are kept for a

minimum of 6 weeks while isolates are stored at -25oC for 5 years.

Analytical failures

In the event of a specimen being unsuitable for processing or where there is an

analytical failure, the laboratory will be informed by phone or in writing.

Discussion of reports

Please contact Prof. M.Cormican at [email protected], Dr. Deirbhile Keady at

[email protected], Dr.Una NiRiain at [email protected], Dr. Eithne McCarthy

at [email protected] , Dr. Teck Wee Boo at [email protected] and Dr.

Dimitar Nashev at [email protected].

Confidentiality Policy

It is the responsibility of all staff, as defined in their contract of employment, to

ensure that all information which they have access to as part of their work is treated in

the strictest confidence and protected from unauthorised access. All staff are asked to

sign a confidentiality agreement during their laboratory induction programme.

Service Level Agreement

The request form for the NSSLRL serves as the formal ‘Service Level Agreement’

between the National Salmonella, Shigella & Listeria Reference Laboratory

[NSSLRL], Diagnostics Directorate, Galway University Hospital (GUH) and the

Service user.

Complaints

Consumer Affairs and the National Advocacy Unit, Quality and Patient Safety

Directorate have responsibility for developing and implementing best practice models

of customer care within the HSE and promotes service user involvement across the

organisation through the concept of “Your Service Your Say”.











Note: If users have a complaint or feel that any part of the service is

unsatisfactory or could be improved on in any way they should contact the

laboratory.

dept of medical microbiology, division of clinical ... · public health ecdc hpsc cdc fsai food...

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