Opportunist Pathogens Unit (OPU)

Services Available

Pathogen	Investigation	Specimen
Acinetobacter spp.	Molecular comparison	Culture
Burkholderia cepacia complex	Identification Molecular comparison	Culture
Burkholderia pseudomallei	Identification	Culture
Burkholderia pseudomallei	Serodiagnosis	Serum
Clostridium difficile	Typing	Culture
Enterobacter spp .	Typing	Culture
Enterococcus spp .	Identification	Culture
Enterococcus spp .	Molecular comparison	Culture
Gram-negative bacteria: non-fermenters only	Identification	Culture
Gram-positive rod (except C.diptheriae)	Identification	Culture
Klebsiella spp .	Typing	Culture
Miscellaneous bacterial species from episodes of hospital infection	Molecular comparison	Culture
Pseudomonas aeruginosa	Typing	Culture
Pseudomonas aeruginosa	Referral criteria	Culture
Pseudomonas aeruginosa	Serodiagnosis	Serum
Stenotrophomonas maltophilia	Molecular comparison	Culture
Serratia spp.	Molecular comparison	Culture

NOTES:
* Cultures should be submitted as pure isolates on agar slopes
* Serum samples should be not less than 200ul.

Epidemiological typing for nosocomial bacterial infections

Capsular typing of Klebsiella spp.

We now offer detection of K1, K2, K5, K54 and K57 capsular types of Klebsiella spp. by multiplex PCR using serotype specific targets in the capsular polysaccharide gene cluster. Putative virulence factors (rmpA and wcaG ) are also sought.

Molecular typing (PFGE) for epidemiological monitoring and surveillance of HealthCare Associated pathogens (see services list)

Currently, molecular comparison of epidemiologically related isolates undertaken in LHCAI is mostly by pulsed-field gel electrophoresis (PFGE). In this method unsheared bacterial DNA prepared in agarose blocks is cut using rare cutting restriction endonucleases to produce, optimally, 20 - 40 fragments. These fragments are then separated through 1.2% agarose in an electric field of 6V/cm alternating in two directions at an incident angle of 120° with the temperature maintained at 12°C and with pulse times increasing from short to long as appropriate for the organisms being compared (see table). Following electrophoresis the banding patterns are stained with ethidium bromide and visualised by ultra violet transillumination. Banding patterns may be interpreted by eye or with the aid of Bionumerics software (Applied Maths, Kortrijk, Belgium).

Organism	Restriction endonuclease	PFGE parameters
Enterococcus spp	SmaI	1 - 35 s pulses for 30 h
Acinetobacter spp	ApaI	5 - 35 s pulses for 30 h
Klebsiella spp	XbaI	5 - 35 s pulses for 30 h
Enterobacter spp	XbaI	5 - 35 s pulses for 30 h
Serratia spp	XbaI	5 - 35 s pulses for 30 h
Pseudomonas aeruginosa	SpeI	1 - 50 s pulses for 30 h
Stenotrophomonas maltophilia	XbaI	5 - 35 s pulses for 30 h

Typing using Variable Number Tandem Repeat (VNTR) analysis, in which isolates are described by the number of repeats found at a number of VNTR loci, is available for some organisms, including Pseudomonas aeruginosa.

Identification of rarely isolated, difficult to identify and atypical gram negative and gram positive aerobic bacteria

This facility is for testing bacteria for which there is no specific reference laboratory. We carry out phenotypic testing and gene sequencing in order to identify both gram-negative fermenters, non-fermenters as well as fastidious gram-negative organisms and gram-positive bacteria. We have identified bacteria from Actinobacillus hominis to Xanthobacter tagetidis.

The main method we use is gas chromatography of cellular fatty acids. A large number of fatty acids and related compounds are known to be present in bacteria. Fatty acids between 9 and 20 carbons in length have been used to characterise bacteria and are particularly useful for the identification of "non fermentative" organisms that are difficult to identify by conventional biochemical tests. The MIDI system detects whole cell fatty acid methyl esters by gas chromatography and by comparing these with an extensive database a wide range of bacteria can be identified.

Burkholderia pseudomallei isolation and identification

Isolation and identification of Burkholderia pseudomallei from specimens of patients suspected of having melioidosis by specialised methods.

PCR identification of Enterococcus spp,Enterobacter sakazakii, Burkholderia spp, P. aeruginosa and S. maltophilia

A speciation and typing service is offered for enterococci. The commoner enterococcal species are tested in a multiplex PCR which will identify E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens and E. avium. Organisms which are negative in this reaction are tested in a panel of 12 biochemical tests.

Burkholderia cepacia -complex isolates are identified by specific PCR and assigned to genomovars following further specific PCRs and, where appropriate, by recA sequence cluster analysis. The gene for cable pili (cblA), associated with the ET12 epidemic strain of genomovar IIIA, is also sought.

A multiplex PCR is available for the identification of P. aeruginosa, S. maltophilia and B. gladioli. PCR identification of Enterobacter sakazakii, based on the ompA gene, is also offered.

Factsheet - Burkholderia cepacia complex and unusual gram negative bacteria from CF sputum

Additional tests on Acinetobacter

In addition to PFGE, the Laboratory carries out PCR to detect bla _OXA carbapenemase genes (in conjunction with ARMRL), class 1 integrase gene (as a marker for outbreak strains), and sequence types corresponding to European clones I, II and III. Identification of A. baumannii by detection of bla _OXA-51-like is carried out routinely on all Acinetobacter isolates. Non-baumannii isolates requiring identification are identified by rpoB sequence cluster analysis. Variable Number Tandem Repeat (VNTR) analysis at two loci is used to provide 'fine-typing' within PFGE defined clusters.

Clostridium difficile

Clostridium difficile isolates can be ribotyped by the Laboratory of Healthcare Associated Infection as per the guidance provided by the CDRNE laboratories. CfI also provides further resolution of ribotype using new molecular methods (joint research with University of Birmingham ).

Pseudomonas aeruginosa serodiagnosis

The serodiagnostic service is based on an in-house ELISA which detects antibodies to the conserved A-band of the lipopolysaccharide of P. aeruginosa.

Pseudomonas aeruginosa referral criteria

Please consult the guidance on environmental and water samples being sent to for molecular analysis of Pseudomonas aeruginosa.

Guidance on samples sent for molecular analysis of pseudomonas aeruginosa (PDF, 331 KB)

Burkholderia pseudomallei serodiagnosis

We regret we have been experiencing technical problems with our current Burkholderia pseudomallei serology test and are therefore suspending this service for the time being. We are currently validating a new test and hope to offer this to customers in the coming months.

Factsheet - Burkholderia cepacia complex and unusual gram negative bacteria from CF sputum
Added/updated: 21 July 2009