FEMS Microbiology Letters 108 (1993) 291-296 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier

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FEMSLE 05383

Lysogenic phage in Salmonella enterica Heidelberg (Salmonella heidelberg): Implications for organism tracing

serovar

Deborah Harvey, Carolyn Harrington 1, Michael W. Heuzenroeder and Christopher Murray

Division of Clinical Microbiology, Institute of Medical and Veterinary Science, Adelaide, S.4., Australia

(Received 9 November 1992; revision received 11 January 1993; accepted 25 January 1993)

Abstract: A phage typing system using a group of 11 closely related phage (as judged by Southern analysis and restriction fragment length polymorphism analysis) was able to distinguish at least six phage types in Salmonella heidelberg of human and animal origin. Restriction fragment length polymorphism analysis using cosmid probes from S. heidelberg confirmed that most S. heidelberg isolates belong to a single 'clonal' group. Southern analysis using DNA isolated from each of the testing phage group showed that phage types 4, 5 and 6 carry closely related endogenous or lysogenic phage. Induction of a lysogenic phage Hlp-4 (Heidelberg lysogenic phage) from type 4 could become lysogenic and convert phage types 1 and 3 to phage type 4 and phage type 5 to a non-typable phenotype.

Key words: Salmonella heidelberg; Bacteriophage type; Restriction fragment length polymorphism; Lysogens; Southern analysis; Phage-type conversion

Introduction

Salmonella enterica serovar Heidelberg (Sal- monella heidelberg) to use the nomenclature of Le Minor and Popoff [1] has emerged as a human pathogen of significance in Australia over recent years and has become firmly established in the human population [2].

Typing of Salmonella for organism tracing has traditionally relied upon serotyping of O (lipo-

Correspondence to: M.W. Heuzenroeder, Division of Clinical Microbiology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia. 1 Present address: State Forensic Science, Divett Place, Ade-

laide 5000, Australia.

polysaccharide) antigen and H (flagella) antigen as developed by Kauffmann and White [3,4]. A limitation of this system is that a relatively small number of S. enterica serovars are responsible for the majority of cases. One commonly used method to subdivide isolates within a serovar is bacterio- phage typing. Different patterns of phage suscep- tibility can be demonstrated both within and be- tween serovars [5-7]. The method can be subjec- tive in nature and is reliant upon the experience of the operator and uniformity of method be- tween laboratories. Recently, molecular methods based upon DNA restriction fragment length polymorphism (RFLP) and multilocus isoenzyme analysis have been shown to be useful and highly sensitive methods for sub-typing these organisms

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[8,9]. Other methods such as plasmid profiling have been shown to be useful in some instances [10] as have a combination of phage typing and RFLP analysis using defined probes such as IS200 for differentiating S. enterica serovar Enteritiditis ( S. enteritidis ) [11].

This paper describes a phage typing system for Salmonella heidelberg and the presence of closely related lysogenic phage in some isolates. These phage could be induced and mediate phage type conversion:

Materials and methods

Materials Agarose was obtained from Sigma and restric-

tion enzymes and T4 ligase from Pharmacia. The nylon membrane, (DIG) labelling and develop- ment kit were obtained from Boehringer-Mann- heim.

Strains and growth conditions S. heidelberg isolates were supplied by the

Australian Salmonella Reference Laboratory, Adelaide, South Australia. All strains were grown in L-broth or on Mueller-Hinton agar plates. The E. coli K-12 strain DH1 was used as host for recombinant cosmids.

Southern analysis RFLP analysis and the derivation of the cos-

mid probe was essentially as reported in [8], DNA was transferred as described by Southern [12] to

nylon membranes. DNA probes, both cosmid and phage, were labelled with Digoxygenin ((DIG)) using random primers and Klenow as per manu- facturer's instructions. The filters were hy- bridized overnight at 42°C in hybridization fluid containing 50% formamide fluid plus (DIG) la- belled probe. The next day filters were washed twice for 10 min at room temperature in 2 x SSC (1 x SSC is 0.15 M NaC1 plus 0.015 M sodium citrate; pH 7) and 0.1% (w/v) SDS and then twice for 20 min at 68°C in 1 x SSC and 0.1% (w/v) SDS. Filters were then developed accord- ing to the manufacturer's instructions.

Phage techniques The group of 11 typing phage was supplied by

the Australian Salmonella Reference Laboratory. Lysogenic phage were induced by UV irradiation for 1 min. Phage were harvested by centrifugation and DNA was extracted according to the method of Maniatis et al. [13]. Phage typing of S. heidel- berg isolates was carried out by the Australian Salmonella Reference Laboratory as previously described [14] using a set of 11 phages isolated and purified in that laboratory using the method described by Adams [15]. Potential lysogens of Hlp-4 were created by streaking 10 ml of high titre > 109 pfu/ml on a dry Mueller Hinton plate. An overnight culture of bacteria was then streaked at right angles to the phage streak. After overnight incubation at 37°C colonies arising in the area of intersection of the two streaks were purified and their DNA extracted. The presence of Hlp-4 sequences was determined by Southern

T a b l e 1

B a c t e r i o p h a g e t y p i n g s y s t e m d e v e l o p e d fo r t he s u b - t y p i n g o f S. heidelberg i s o l a t e s

P h a g e type S e n s i t i v i t y to t h e p h a g e t y p i n g g r o u p

1 2 3 4 5 6 7 8 9 10 11

N u m b e r o f i so l a t e s *

1, + + - + - + + - + + - 48

2 + . . . . + - - + + - 20

3 + + + + + + + + + + + 1

4 . . . . . . . . . + + 4

5 + - - + - + - - + + - 9

6 - + - + - - + . . . . 1

+ , s ens i t i ve ; - , r e s i s t a n t .

* I n c l u d e s two t h a t w e r e p h a g e - s e n s i t i v e , b u t d o n o t c o n f o r m to a r e c o g n i s e d p a t t e r n a n d f o u r w e r e u n t y p a b l e .

analysis using purified panel phage 2 DNA as a probe as described above.

Outer membrane studies SDS polyacrylamide gel electrophoresis,

Coomassie brilliant blue [16] and silver staining for LPS and outer membrane profiles [17] was as described previously [18].

Results and Discuss ion

S. heidelberg phage typing scheme The group of 11 phage could distinguish six

groups within S. heidelberg. A total of 89 isolates were examined. The phage typing scheme is de- picted in Table 1. Phage types 1 and 2 are the predominant types consisting of 50 and 20 iso- lates, respectively. Most isolates (68/89) of all phage types were from humans and (16/17) of chicken isolates were phage type 1. The remain- ing four isolates were from other sources but were predominantly (3/4) phage type 1 [2].

RFLP analysis using S. heidelberg cosmid DNA RFLP analysis of HindlII-digested chromoso-

mal DNA shows that the six S. heidelberg phage types, with the exception of the rare type 6 (1/89) which has a related pattern have the same RFLP pattern using the cosmid plMVS 100 (Fig. 1). Similar results were also seen using the indepen- dently isolated cosmid plMVS 101 and cosmids derived from S. enterica II serovar Sofia (S. sofia) (data not shown). These data confirm the results of a multi-locus enzyme analysis [9] which sug- gests that the majority of S. heidelberg isolates belong to a single 'clonal' grouping and that serovars not conforming to the clonal genotype are the result of the genetic exchange of the rfb locus which specifies the O serovar. Genetic ex- change within the rfb gene cluster in S. enterica has occurred independently many times [19].

Southern analysis using typing panel bacteriophage DNA

Initial Southern hybridization analysis sug- gested that the phage used in the typing panel were related to each other since they cross lay-

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MW 6 5 0 5 4 3 0 3 2 lO 1

8

6

2.'l

Fig. 1. Southern hybridization analysis depicting the RFLP patterns obtained after a H/ndlII digestion of each of the six phage types and probed with (DIG) labelled plMVS 100. Tracks labelled 1-6 contain phage types 1-6, respectively; the asterisk (*) indicates Hip-4 lysogens. MW is the molecular size marker (EcoRI-digested SPP1), sizes are given in kilo-

bases.

bridize at relatively high levels of stringency (1 × SSC, 0.1% SDS 68°C; data not shown). When phage DNA from any of the 11 typing phage were used to probe-digested genomic DNA from the six S. heidelberg phage types, identical RFLP patterns were obtained regardless of which of the 11 typing panel phage were used. A typical result using typing panel phage 2 as a probe is shown in Fig. 2. This shows the presence of sequences of related lysogenic or endogenous phage within S. heidelberg phage types 4, 5 and 6. The RFLP patterns of these phage are distinct from panel phage 2. The common bands at 4.5 kb and 2 kb may represent hybridization to host chromosomal sequences carried by the probing phage that may be involved in the integration of the phage into the host chromosome. It is also possible that the lysogenic phage may not be closely related per se to the panel of typing phage, but hybridization of common fragments may be due to the carriage by both phage of IS elements such as IS200 which is reported to be Salmonella-specific [11].

Induction of putative lysogenic phage Phage were induced from S. heidelberg phage

types 2, 4, 5 and 6. These were designated Hlp-

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6 5 ~ 4 30 3 2 10 1 P2 MW

B

6

4.8

2.7

1.9

Fig. 2. Southern hybridization of a HindlII digest of genomic DNA of each phage type. When probed with (DIG) labelled typing panel phage DNA, an identical pattern to this figure was always obtained regardless of the phage DNA used. The confirmation of the presence of Hip-4 sequences in phage types 1, 3 and 5 after lysogeny is indicated by an asterisk (*) adjacent to the number indicating the phage type (1-6). The H/ndlII digested genomic DNA from each phage type was probed using (DIG) labelled typing phage 2. MW indicates molecular sizes in kilobases based upon EcoRI-digested SPP1

DNA.

(Heidelberg lysogenic phage) 2, 4, 5 and 6, re- spectively. With the exception of Hlp-2, these phage appeared to be related to each other on the basis of RFLP analyses (not shown). The ability of these induced phage to plaque on the six S. heidelberg phage types is shown in Table 2. Phage type 6 appears to be resistant to all four Hip phage; Hip-5 can plaque on its host strain which suggests that no immunity to super-infec- lion to Hip-5 is conferred by the carriage of lysogenic Hlp-5.

S. heidelberg phage type conversion using phage Hlp-4

Phage Hip-4 was used to make lysogens in other S. heidelberg phage types. It was possible to

demonstrate the presence of Hlp-4 sequences in phage types 1, 3 and 5 (Fig. 2). Phage type 5 appeared to allow super-infection with Hip-4, since sequences of Hip-5 and Hlp-4 could both be detected in Southern analysis. It is also consistent with Hip-5 not mediating immunity to super-in- fection with itself (Table 2). It was not possible to create lysogens in phage types 2 or 6, both of which carry endogenous phage and presumably mediate resistance to super-infection with their lysogenic phage or other related exogenous phage. The lysogens created by Hlp-4 were then tested to determine if phage type conversion had oc- curred. Lysogeny with Hip-4 converted phage types 1 and 3 to phage type 4 and phage type 5 was converted to a non-typable strain. Analysis of outer membrane proteins and LPS did not reveal differences in LPS or outer membrane protein profiles between phage types or strains created by phage type conversion (data not shown).

Conclusion Bacteriophage typing is a well-established

method for strain identification and tracing of organisms for epidemiological purposes. Phage type can also be a virulence marker in some species. In S. enteritidis, the conversion of phage type 4 to phage type 7 results in a modification of lipopolysaccharide together with a loss of Viru- lence [20]. Lysogeny in S. enterica serovar Choler- aesuis (S. choleraesuis) by phage 14 has been reported to result in increased O-antigen chain length, serum resistance and virulence in a mouse model [21]. Lysogeny by Hlp-4 in S. heidelberg did not result in any detectable outer membrane or LPS changes.

Table 2

Bacteriophage resistance patterns of S. heidelberg phage types when tested against (Hlp)-induced phage from phage types 2, 4, 5 and 6

Phage S. heidelberg phage type

1 2 3 4 5 6

Hip-2 + - + + + Hip-4 + + + - + Hip-5 + + + + + Hip-6 + + + + +

The p h a g e type of an o rgan i sm can be de te r - m i n e d by the ca r r i age o f lysogenic phage , or the p re sence o r absence of p h a g e r ecep to r s u p o n the cell surface. T h e da t a in this p a p e r i l lus t ra te po ten t i a l p r o b l e m s in the e m p l o y m e n t of p h a g e typing a lone for cr i t ical o rgan i sm t rac ing where convers ion o f one p h a g e type to ano the r is read i ly accompl i shed by lysogeny. T h e f requency of such convers ion in vivo is ye t to be d e t e r m i n e d . Classi- cal m e t h o d s such as p h a g e typing will r e m a i n useful tools for the ep idemio log i s t c o m p l e m e n t e d with the newer m e t h o d s of R F L P and mul t i - locus i soenzyme analysis.

Acknowledgement

W e would l ike to acknowledge the gene rous s uppo r t of the A u s t r a l i a n Chicken M e a t Re- search and D e v e l o p m e n t Counci l .

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