Opportunist Pathogens Section

Epidemiological typing for nosocomial bacterial infections

Phenotypic schemes are available for Pseudomonas aeruginosa (O serotyping), Klebsiella spp. and Enterobacter aerogenes (K serotyping) and Enterobacter cloacae (O serotyping, biotyping).

O (lipopolysaccharide) and K (capsular) serotyping allow the assignment of definitive types. Isolates with different serotypes can, with confidence, be assumed to represent unrelated strains. However, 'non-typable', polyagglutinable' or 'autoagglutinable' strains cannot be considered as distinct types since they often result from the loss of the surface structures responsible for defining serotype. Unless the serotype is uncommon, identity of serotype is insufficient evidence for defining a single strain. Additional methods such as molecular comparison by pulsed field gel electrophoresis (PFGE) may be used to distinguish strains.

We now offer detection of K1, K2 and K5 serotypes of Klebsiella spp. by multiplex PCR using serotype specific targets in the capsular polysaccharide gene cluster. PCR identification of Enterobacter sakazakii, based on the ompA gene, is also available.

Biotyping suffers from poor reproducibility but biotypes can be useful for identifying unusual strains of Enterobacter cloacae.

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 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
Staphylococcus aureus	SmaI	1 - 80 s pulses for 30 h
Coag. neg. staphylococci	SmaI	1 - 45 s pulses for 30 h
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

Identification of gram negative non fermenting rods and gram positive aerobic rods

Identification by gas chromatography of their 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., 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 RFLP of the amplicon and confirmation by further specific PCRs and, where appropriate by recA sequence cluster analysis. PCR tests are offered for the putative transmissibility factors of epidemic Burkholderia cepacia genomovar IIIa from CF patients (cable pili, insertion sequence hybrid and epidemic strain marker gene).

A multiplex PCR is available for the identification of P. aeruginosa, S. maltophilia and B. gladioli.

 

Additional tests on Acinetobacter

In addition to PFGE, the Laboratory also 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.

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 ).

Last reviewed: 4 September 2008