Diversity of bacteriophages encoding Panton-Valentine leukocidin in temporally and geographically related Staphylococcus aureus.

Production of the Panton-Valentine leukocidin (PVL) by Staphylococcus aureus is mediated via the genes lukS-PV and lukF-PV which are carried on bacteriophage ϕSa2. PVL is associated with S. aureus strains that cause serious infections and clones of community-associated methicillin-resistant S. aureus (CA-MRSA) that have additionally disseminated widely. In Western Australia (WA) the original CA-MRSA were PVL negative however, between 2005 and 2008, following the introduction of eight international PVL-positive CA-MRSA, PVL-positive WA CA-MRSA were found. There was concern that PVL bacteriophages from the international clones were transferring into the local clones, therefore a comparative study of PVL-carrying ϕSa2 prophage genomes from historic WA PVL-positive S. aureus and representatives of all PVL-positive CA-MRSA isolated in WA between 2005 and 2008 was performed. The prophages were classified into two genera and three PVL bacteriophage groups and had undergone many recombination events during their evolution. Comparative analysis of mosaic regions of selected bacteriophages using the Alignments of bacteriophage genomes (Alpha) aligner revealed novel recombinations and modules. There was heterogeneity in the chromosomal integration sites, the lysogeny regulation regions, the defence and DNA processing modules, the structural and packaging modules and the lukSF-PV genes. One WA CA-MRSA (WA518751) and one international clone (Korean Clone) have probably acquired PVL-carrying ϕSa2 in WA, however these clones did not disseminate in the community. Genetic heterogeneity made it impossible to trace the source of the PVL prophages in the other WA clones. Against this background of PVL prophage diversity, the sequence of one group, the ϕSa2USA/ϕSa2wa-st93 group, was remarkably stable over at least 20 years and associated with the highly virulent USA300 and ST93-IVa CA-MRSA lineages that have disseminated globally.

Introduction

Staphylococcus aureus is a pandemic pathogen that is also part of the human microbiota [1]. Paramount to the success of S. aureus has been its ability to utilize mobile elements to acquire and disseminate antibiotic resistance, virulence and adaptive mechanisms amongst staphylococcal populations. In methicillin-sensitive S. aureus (MSSA) and community-associated methicillin-resistant S. aureus (CA-MRSA) the Panton-Valentine leukocidin (PVL) is a virulence factor that is carried on a bacteriophage known as ϕSa2 which is integrated into the chromosome as a prophage [2]. PVL is a bi-component, pore-forming toxin produced by co-transcribed genes, lukF-PV and lukS-PV, that targets and lyses human macrophages, polymorphonuclear leukocytes and monocytes and also incites the human inflammatory immune response [3]. Strains encoding PVL are associated with skin and soft tissue infections and dangerous invasive infections however, the role that the toxin plays in virulence is controversial and as yet, no clear-cut selective advantage has been shown for CA-MRSA that produce PVL [4-8]. Many virulent strains of MSSA and CA-MRSA do not produce PVL, however, as the pathogen evolves it is evident that those that have disseminated to cause the greatest burden of infectious disease harbor the prophage [9].

The PVL bacteriophage genome is composed of functionally colinear main modules encoding genes for lysogeny, DNA processing, head morphogenesis and packaging, tail morphogenesis, and lysis, with the lukSF-PV genes encoded between the lysis and lysogeny modules in the circularly permutated bacteriophage [10]. The lysogeny, lukSF-PV and lysis regions are well conserved with minor polymorphisms. Most diversity occurs in the DNA processing module with the head and tail morphogenesis genes showing diversity depending on the genus and PVL group. ϕSa2 can be vertically transmitted with the chromosome during replication or it can enter the lytic cycle and transmit horizontally to another cell. It has been well documented that bacteriophages undergo high rates of recombination and both these forms of transmission allow opportunity for genetic exchange, the potential mechanisms being transposition, site-specific recombination, homing endonucleases and homologous and illegitimate recombination [11]. While it is believed that horizontal gene transfer between S. aureus of different lineages is rare due to a lineage-specific type1 restriction-modification system [12], an in-vivo study revealed that bacteriophage transferred frequently during co-colonization by S. aureus of the same lineage and recombination between different bacteriophage occurred [13]. An investigation of MRSA colonisation in remote WA revealed that 8% of screening swab sets with an MRSA were colonised with multiple lineages of MRSA and 51.7% were co-colonised with an MSSA [14]. This would provide ideal opportunities for bacteriophage transmission and recombination to occur.

Five lineages of CA-MRSA, ST1-IVa (WA1), ST78-IVa (WA2), ST5-IVa (WA3), ST45-V (WA4) and ST8-IVa (WA5) emerged in remote Western Australian (WA) communities and WA1, WA2 and WA3 eventually disseminated to the capital city Perth and the eastern states of Australia [15, 16]. Unlike CA-MRSA that were being reported outside of WA, the WA strains were PVL negative [17]. There were however, two lineages of PVL-positive MSSA in remote WA communities, ST93-MSSA and ST121-MSSA [14].

In 2005, a PVL-positive strain belonging to the same lineage as WA1 was isolated, followed in 2008, by WA2-, WA3- and WA5-like PVL positive clones. In WA, all MRSA are submitted to a central facility for typing and epidemiological investigation [18] and between 2005 and 2008 eight international PVL-positive CA-MRSA were introduced into WA. The rise in the number of PVL-positive CA-MRSA in WA since the first was found in 2003 has been alarming. In 2003/2004 2.1% of CA-MRSA were PVL positive, however by 2015/2016 this had risen to 52.8%, with the predominant clones being ST93-IVa (Queensland clone, 63%), ST5-IVc (WA 121, 19.5%) and ST30-IVc (WSPP, 6.8%). WA1-, WA2- and WA3-like PVL-positive clones were still in the community in 2016 however, they had not thrived and formed lower percentages of 0.7%, 0.17% and 1.1% respectively while PVL-positive WA5 had disappeared [19].

The overall aims of this study were to investigate PVL prophages from lineages of PVL-positive MRSA isolated in WA between 2005 and 2008 firstly, to gain insights into the genetics of geographically and temporally related PVL prophages in WA and secondly, to determine if PVL bacteriophages from the international strains had horizontally transmitted into the local WA clones. A comparative analysis of the PVL prophages has been performed using conventional sequence analysis, and regions of selected prophages have been compared using the Alignments of bacteriophage genomes (Alpha) aligner, which is an application that creates a partial order of gapless alignments along the bacteriophage genomes, allowing the identification of common core sequences and modular segments [20, 21]. Heterogeneity between the bacteriophages has been investigated using Alpha aligner defined modules and coding sequence comparisons. PVL bacteriophages were induced from PVL-positive S. aureus from the WA community and attempts were made to lysogenise prototype PVL-negative WA CA-MRSA.

Materials and methods

Bacterial strains

Genotypes and year of isolation of PVL-positive clones and their PVL prophage sizes are presented in Table 1. All MRSA except WA2RNSH95 and USA300 FPR3757 were from cases of infection or colonization in the WA community [18]. WA2RNSH95 was a WA2 clone from Sydney, Australia. The USA300 clone was present in WA [22] and the prophage ϕSa2USA from FPR3757 (Genbank: NC_007793) was used for genetic comparison. MSSA isolates W17S and K25S were colonizing isolates from remote WA communities [14]. ST772-V was previously sequenced [23]. MW2 (Genbank: BA000033) was used as a lukSF-PV gene-sequencing and prophage induction control. ϕSLT (Genbank: AB045978) and ϕSa2958 (Genbank: AP009363) were lukSF-PV gene sequencing controls.

Table 1

WA PVL-positive bacteriophages and lysogens.

Bacteriophage	Size (bp)	Lysogen genotypeCC, ST-SCCmec	CloneStrain	Year of isolation	Reference
WA PVL-positive Clones
ϕSa2wa-st1	45,585	1, ST1-IVa	WA115798	2005	This study
ϕSa2wa-st5	44,823	5, ST5-IVa	WA318790	2008	This study
ϕSa2wa-st8	45,914	8, ST8-IVa	WA518751	2008	This study
ϕSa2wa-st78	45,878	88, ST78-IVa	WA2RNSH95	2008	This study
ϕSa2wa-st93mssa	45,913	Singleton, ST93	W17S	1995	[14]
ϕSa2wa-st121mssa	45,621	121, ST121	K25S	1995	[14]
International Clones
ϕSa2wa-st22	38,576	22, ST22-IVc	16386	2007	[18]
ϕSa2wa-st30	45,780	30, ST30-IVc	WSPP16663	2002	[25]
ϕSa2wa-st59	42,133	59, ST59-V	Taiwan clone16672	2003	[26]
ϕSa2wa-st72	47,213	72, ST72-IVa	Korean clone15803	2006	[18]
ϕSa2wa-st80	45,164	80, ST80-1Vc	European clone15395	2004	[27]
ϕSa2wa-st93	45,913	ST93-IVa	Qld clone16790	2003	[28]
ϕSa2wa-st772	42,402	1, ST772-V	Bengal Bay clone17048	2007	[23]
ϕSa2USA	45,914	8, ST8-IVa	USA300_FPR3757	2003	[29]
WA PVL-negative Clones
NA	NA	1, ST1-IVa	WA1WBG8287	1995	[24]
NA	NA	88, ST255-IVa	WA2WBG8366	1995	[24]
NA	NA	5, ST5-IVa	WA3WBG8378	1995	[24]
NA	NA	45, ST45-V	WA4WBG8404	1995	[24]
NA	NA	8, ST8-IVa	WA5WBG7583	1989	[30]

Abbreviations: bp, base pairs; NA, Not applicable; WA, Western Australian, Qld, Queensland; WSPP, Western Samoan Phage Pattern

PVL-negative WA1WBG8287, WA2WBG8366, WA3WBG8378, WA4WBG8404 and WA5WBG7583 are historic prototype clones from the WA community [24]. Bacteriophage indicator and propagating strains were RN4220, WBG248, WBG356, WBG696 and WBG286.

Sequencing of bacterial and PVL-prophage genomes and genetic analysis

Twelve bacterial genomes were sequenced using Illumina NextSeq sequence chemistry (Illumina Australia, Scoresby, Victoria 3179) and assembled with SPAdes, v3.9.0. The PVL-prophage reads were extracted and analysed using MacVector with Assembler, v15.5.3 (Accelrys, Cambridge, UK). The sequences of ϕSa2wa-st1, -st8, -st30, -st72 and -st93mssa were on single contigs, the remainder were assembled by overlapping contigs utilising the MacVector Assembler bowtie and phrap algorithms. Bacteriophage were designated as phi Sa2 Western Australia-host sequence type (ϕSa2wa-st). Except for prophages ϕSa2USA and ϕSa2wa-st772, National Centre for Biotechnology Information (NCBI) homology searches used only whole bacteriophage genome sequences for comparisons.

lukSF-PV sequencing

Isolates were cultured on brain heart infusion agar (BHIA) (Gibco Diagnostics, Gaithersburg, MD, USA), incubated at 37°C, grown in trypticase soy broth (Gibco Diagnostics, Gaithersburg, MD, USA) and incubated overnight at 37°C. DNA was extracted using the Invitrogen PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer with lysostaphin (Sigma-Aldrich, St. Louis, MO, USA) used to lyse the S. aureus cell wall. lukSF-PV was amplified as previously described [31]. Amplicons were purified using the Ultraclean DNA PCR Clean Up Kit (MoBio Laboratories, GeneWorks, Thebarton, SA, Australia) and sequences were compared with the lukSF-PV genes from ϕSLT (Genbank: AB045978).

Bacteriophage induction and hybridisation

Bacteriophage were induced using Mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) as previously described [10]. Plaques were transferred onto nylon membranes using standard techniques [32] and DNA was cross-linked to the membrane (Amersham Biosciences, Little Chalfont, Bucks, England) using a GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were treated with 2 mg/mL Proteinase K (Roche Diagnostics, Mannheim, Germany). The hybridisation probe was obtained by PCR amplification of lukSF-PV using previously described primers [33]. PCR products were purified using the MoBio PCR Cleanup Kit. Probes were prepared using the DIG DNA Labelling and Detection Kit according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). Plaque hybridisation was performed as directed by the manufacturer (Boehringer Mannheim, Mannheim, Germany).

PVL-bacteriophage propagation and lysogenisation of PVL-negative WA CA-MRSA

To propagate the mitomycin C-induced PVL-positive bacteriophages the plaques were extracted and crushed with 3 drops of BHIB, and the mixture left to stand for 10 minutes. This suspension was added to 100 μL of an overnight culture of the indicator strain, 3 mL of molten 3% BHIA was added and the mixture poured onto a BHIA plus 0.004M Ca2+ base plate which was incubated overnight at 30°C. The overlay containing the bacteriophage and indicator strain was scraped off and filtered. Each of the PVL-negative strains of WA CA-MRSA were grown overnight in BHIB and lawn-inoculated onto BHIA plus 0.004M CaCl2. A drop of each PVL-bacteriophage lysate was placed on the lawn and incubated at 30°C overnight. Isolated colonies growing in the centre of plaques present on lawns of PVL-negative WA clones were picked, their total DNA was isolated and lysogeny was detected using previously described primers [33].

Results

Sequence analysis and bacteriophage classification reveal diversity amongst the prophages

Fourteen prophage genomes between flanking direct 21 base pair (bp) repeats of 5′-AGGGCAAAAAAAGGGCg/aGATT-3′ termed attL and attR were analysed (Table 1). The 12 new prophage sequences from this study have been deposited in the NCBI database under accession numbers MF580410, MK940809 and MG029509 to MG029518.

The prophages were between 38,576 and 47,213 bp in size with between 41.4 and 100% nucleotide (nt) identity, GC compositions of 31 to 33.4% and 52 to 75 protein-coding sequences of 25 or more amino acids (aa).

The prophage genomes had the organisation of Siphoviridae family Sfi21-like PVL viruses of the Caudovirales order and, according to the most recent staphylococcal bacteriophage classification criteria, were placed into two genera and three PVL bacteriophage groups (Table 2) [34-36]. ϕSa2wa-st22, -st59 and -st772 (76.1–76.7% nt identity) were placed into the 77likevirus genus of icosahedral-headed bacteriophage. ϕSa2wa-st22 and -st772 were group 1 PVL bacteriophage with 74.4% nt identity and ϕSa2wa-st59 was group 3. ϕSa2wa-st1, -st5, -st8, -st30, -st72, -st78, -st80, -st93, -st93mssa, -st121mssa and ϕSa2USA (74.2–100% nt identity) were 3alikevirus genus, prolate-headed group 2 PVL bacteriophage. ϕSa2wa-st5 was unusual in that it encoded type C DNA polymerase (Genbank: AUM57702) rather than type A (exemplified by ϕSa2wa-st93 Genbank: AUM58245) (Fig 1).

Table 2

WA PVL prophage lukSF-PV polymorphisms, prophage classifications and lysogen lineages.

Prophage	LysogenCC, ST	Lysogen Genus/PVL gp.	SNPs
			lukS-PV						lukF-PV
			33	105	345	443	527	663	1186	1396	1729
ϕSLT	30, ST30	3alikevirus/2	G	T	C	G	A	G	C	A	A
ϕSa2wa-st30	30, ST30	3alikevirus/2	G	T	C	G	A	G	C	A	A
ϕSa2wa-st772	1, ST772	77likevirus/1	G	T	C	G	A	G	C	A	A
ϕSa2958	5, ST5	3alikevirus/2	G	T	C	G	A	G	C	G	A
ϕSa2wa-st1	1, ST1	3alikevirus/2	G	T	C	G	A	G	C	G	A
ϕSa2wa-st22	22, ST22	77likevirus/1	G	T	C	G	A	G	C	G	A
ϕSa2wa-st59	59, ST59	77likevirus/3	G	T	C	G	A	G	C	G	A
ϕSa2wa-st8	8, ST8	3alikevirus/2	G	T	C	G	G	T	C	A	G
ϕSa2wa-st72	72, ST72	3alikevirus/2	G	T	C	G	G	T	C	A	G
ϕSa2wa-st93	S, ST93	3alikevirus/2	G	T	C	G	G	T	C	A	G
ϕSa2wa-st93mssa	S, ST93	3alikevirus/2	G	T	C	G	G	T	C	A	G
ϕSa2USA	8, ST8	3alikevirus/2	G	T	C	G	G	T	C	A	G
ϕSa2wa-st5	5, ST5	3alikevirus/2	G	T	C	A	A	G	C	G	A
ϕSa2wa-st78	88, ST78	3alikevirus/2	G	C	C	G	A	G	C	G	A
ϕSa2wa-st121mssa	121, ST121	3alikevirus/2	G	T	C	G	A	G	T	A	A
ϕSa2wa-st80	80, ST80	3alikevirus/2	A	T	T	G	A	G	C	A	A
ϕSa2mw	1, ST1	3alikevirus/2	G	T	C	G	G	T	C	A	A

Nucleotides differing from those of ϕSLT are shaded. Abbreviations: gp., group

Fig 1

Diagrammatic comparison of ϕSa2wa-st5 with ϕSa2wa-st1 and ϕSa2wa-st93.

ϕSa2wa-st5 ORFs are represented as arrows indicating the direction of transcription and coloured according to the PVL prophage or groups of PVL prophages from PVL-positive S. aureus in WA that share 97 to 100% nucleotide identity. Where identity is less than 97% this is indicated under the ORFs. Regions of the genomes with 97 to 100% identity with ϕSa2wa-st5 are shaded. Where identity is less than 97% this is indicated in the shaded region. The main functional modules are indicated on a line above the genomes. Red flags indicate attL and attR sites. Genome size is indicated at the right-hand end. Proteins encoded by ORFs relevant to this study and structural proteins are indicated. Hypothetical proteins are identified by their Genbank accession number. The positions and sequences of DR4 and a widely-shared recombination site are presented. Abbreviations: DR, direct repeat; HP, hypothetical protein.

ϕSa2wa-st93, -st93mssa (100% nt identity) and -st8 (99.97% nt identity) were considered to be the same bacteriophage as the international ϕSa2USA (99.97% nt identity), with ϕSa2wa-st72 (96.6% nt identity) very closely related. These will be known as the ϕSa2USA/ϕSa2wa-st93 group in this study. The prophages found in the WA clones, WA115798 (ϕSa2wa-st1), WA2RNSH95 (ϕSa2wa-st78) and WA318790 (ϕSa2wa-st5) had identities of 80.8 to 93.8% and, although related, they were not identical to each other or any PVL bacteriophage in this study or in the NCBI database while ϕSa2wa-st8 from WA518751 had only 1 bp difference with ϕSa2USA and will be included in the ϕSa2USA/ϕSa2wa-st93 group.

The ϕSa2 chromosomal integration site was heterogenous

Chromosomal sequences proximal to the prophage terminals encoded the hybrid attLi and attRi sites of the attB and attP sites on the chromosome and a circularly permuted form of the bacteriophage. They consist of a 29-bp central core and 25-bp left-hand (LH) and right-hand (RH) arms (Fig 2) [37]. There were nine single nucleotide polymorphism (SNP) profiles for attLi and seven for attRi (Fig 2). Two groups of prophages shared identical attLi and attRi sites; international prophage ϕSa2USA and ϕSa2wa-st72 with ϕSa2wa-st8, and Australian international prophage ϕSa2wa-st93 with ϕSa2wa-st93mssa. Of the prophage in the WA CA-MRSA-like strains, ϕSa2wa-st8 shared attRi with the ϕSa2USA/ϕSa2wa-st93 group and ϕSa2wa-st772; ϕSa2wa-st1 had a unique attLi and ϕSa2wa-st8 shared attLi with ϕSa2wa-st72 and ϕSa2USA. ϕSa2wa-st5 and ϕSa2wa-st78 had unique integration-site sequences. attLi of ϕSa2wa-st78 could not be identified, however its attRi was reasonably similar to the ϕSa2wa prophages over the LH arm and the first 17 bp of the common core (3 bp difference) while 32 of the remaining 37 bp were different (Fig 2). attLi of ϕSa2wa-st30 was absent due to a 268 bp deletion (detected by comparison with the intact “preferred integration site” of WA2RNSH95).

Fig 2

Integration-site sequences proximal to the terminals of PVL prophages from PVL-positive S. aureus in WA (2005 to 2008).

Sequences have been aligned using ClustalW. The central core sequence is underlined with a thick black line. Left-hand and right-hand arms are indicated. SNP profiles are numbered alongside their respective bacteriophages. Identical nucleotides are indicated by an asterisk, absence of an asterisk indicates a polymorphic site. Abbreviations: LH, left hand; RH, right hand; attLi, left-hand integration site; attRi, right-hand integration site.

With the exception of ϕSa2wa-st78, the bacteriophages had inserted into a gene within a cluster of three or four open reading frames (ORFs) encoding a putative domain of unknown function (DUF)1672 lipoprotein [38], one downstream of the integration site and two or three upstream. The four DUF1672 domain-containing proteins of ϕSa2wa-st72 had amino acid similarity scores of 61.1–79.7% indicating they were paralogues. There was variability in the truncated ORF. ϕSa2wa-st1, -st5, -st59, -st772 and the ϕSa2USA/ϕSa2wa-st93 group had truncated the 3' end of an ORF encoding a lipoprotein_7 superfamily domain-containing protein (54.5–100% nt identity and 55.6–100% amino acid similarity) which variably also encoded a structural maintenance of the chromosome SMC_N domain (ϕSa2wa-st1, -st5, -st59 and -st72). ϕSa2wa-st22, -st30, -st80 and -st121mssa, had truncated an ORF encoding a hypothetical protein (HP) which was in the same position as the lipoprotein_7 domain ORF but lacked the lipoprotein_7 domain. ORFs truncated by ϕSa2wa-st22, -st30 and -st80 had 82.2–94% nt identity however, the ORF truncated by ϕSa2wa-st121mssa had only 26.4–36.2% nt identity. Immediately upstream of all prophages except ϕSa2wa-st78 was an ORF encoding a 62-aa HP whose sequence indicated that it was the 3' terminal of the truncated lipoprotein_7 protein ORF, when compared with the intact lipoprotein_7 domain-encoding ORF of WA2RNSH95 (not shown).

ϕSa2wa-st78 had truncated the 3' end of a 6-phospho-beta-galactosidase gene and inserted upstream of a galactose-6-phosphate degradation enzyme. The ϕSa2wa-st78 host genome, WA2RNSH95, encoded an intact lipoprotein_7 domain-encoding ORF that contained an attLi site which was homologous over the LH arm and central core (1 bp difference) with the consensus attLi but had 14 bp differences in the RH arm. This may have prevented insertion of ϕSa2wa-st78 into what appears to be a preferred site for ϕSa2.

Lysogeny regulation and modular recombination sequences

The intergenic region between the divergently transcribed integrase gene int and its associated HP ORF, originally called orfC [39] (Fig 1), contained structures indicative of involvement in regulation and lysogeny in all prophages (Fig 3). There were SNPs between the prophages, however all except ϕSa2wa-st772 had the same secondary structure which consisted of a consensus sigma factor H (SigH) binding-site [40] and a downstream inverted repeat (IR) of 5'-GAACGTAc/tGTTC-3'. Overlapping the SigH binding-site was an inverted repeat that could form a possible stem-loop structure of 5'-GGGTAGgtgggCTACCC-3' (stem-loop 1) (Fig 3). The first two nucleotides of the loop could be GT, TC or GC (ϕSa2wa-st772). There was then a previously identified and highly conserved stem-loop putative regulatory site, stem-loop 2 [41]. Both stem-loops were flanked by heptanucleotide direct repeats (DR) of 5'-AAAATAA-3 (DR1) the first of which comprised 7 bp of the SigH binding site.

Fig 3

Regulation regions of PVL prophages from PVL-positive S. aureus in WA (2005 to 2008).

Sequences have been aligned using ClustalW. The SigH binding site is shaded. Identical nucleotides in the alignment are indicated by an asterisk. Repeats are indicated by arrows. Abbreviations: IR = inverted repeat; DR = direct repeat.

ϕSa2wa-st772, which has previously been predicted to be a recombinant bacteriophage [23] had a regulation region that was somewhat different. The intergenic region was between int and a different HP ORF (exemplified by YP_00910342) transcribed on the same strand. The regulatory features however, included the SigH binding-site with its downstream IR and stem-loop 1; stem-loop 2 was absent and there was only one copy of DR1, which occurs from 24 to 33 times in the prophage genomes.

Of the prophages in the WA CA-MRSA the ϕSa2wa-st1 regulation region was identical with that of ϕSa2wa-st80 while ϕSa2wa-st5, -st8 and -st78 were identical with ϕSa2USA.

A previously described 23-bp recombination site that has been found in unrelated staphylococcal bacteriophage [42] was found downstream of the holin gene in all prophages (Fig 1). ϕSa2wa-st772 encoded the enterotoxin A gene flanked by direct repeats (DRs) of 5'-CTTTTTATTTTG-3' immediately downstream of this site thus implicating the site in the acquisition of an extra virulence factor, probably from an unrelated family ϕ3 beta haemolysin-converting bacteriophage.

ϕSa2wa-st5 has a module of 592 bp (bp 5,327–5,918) which encodes a DUF2829 protein and a HP ORF (Fig 1, Genbank: AUM57690 and AUM57691) flanked by 42-bp direct repeats (Fig 1, DR4). This module is present in ϕSa2wa-st1, -st30, -st78, -st80 and -st121mssa however, ϕSa2wa-st1, -st80 and -st121mssa lack a LH copy of DR4. Furthermore, this repeat is present as a similarly positioned single copy in all other study prophage except ϕSa2wa-st22 and -st772, indicating that the module has disseminated horizontally between bacteriophage of the same genera and the repeat is a conserved sequence that could mediate recombination, integration and excision.

lukS/F-PV sequence SNPs were not specific for PVL bacteriophage genera or lysogen genotype

A single copy of a DR that has been implicated in the deletion of the lukSF-PV and integrase module [43] was found downstream of the lukSF-PV genes in all prophages however, a second copy was not be found in any of the genomes. Nine lukSF-PV SNPs were found, six in lukS-PV and three in lukF-PV and there were eight SNP profiles (Table 2). All except the A→G (histidine→arginine) substitution at position 527 in the ϕSa2USA/ϕSa2wa-st93 group and ϕSa2MW, were synonymous. The ϕSa2USA/ϕSa2wa-st93 group SNPs were identical. ϕSa2wa-st30 and -st772 were identical to ϕSLT. ϕSa2wa-st22, -st59 and -st1 were identical to the CC5 control ϕ2958. ϕSa2wa-st78, -st80 and -st121mssa had individual lukSF-PV SNPs reported previously for their respective genetic lineages [44, 45] and ϕSa2wa-st5 had a unique lukSF-PV SNP profile.

The distribution of the SNP profiles was heterogenous. (i) Highly similar PVL prophage with the same lukSF-PV SNPs lysogenised S. aureus of three different lineages, indicating horizontal dissemination of a successful PVL bacteriophage between S. aureus of three lineages; the ϕSa2USA/ϕSa2wa-st93 group lysogenised ST8-IVa, ST72-IVa, ST93, and ST93-IVa. (ii) Different PVL prophages with different lukSF-PV SNP profiles lysogenised S. aureus of the same lineage, indicating horizontal transmission of different PVL bacteriophage into S. aureus of the same lineage; 77likevirus, PVL group 1 (ϕSa2wa-st772) and 3alikevirus, PVL group 2 (ϕSa2wa-st1 and ϕSa2mw) prophages lysogenised CC1 strains, ST772-V and ST1-IVa respectively. (iii) Different genera of PVL prophage with the same lukSF-PV SNP profile lysogenised different S. aureus lineages, indicating that different PVL bacteriophage can carry the same lukSF-PV module and that either the lukSF-PV genes disseminate horizontally between different genera of PVL bacteriophage or random substitutions occur during replication and the fittest permutations prevail regardless of the genus of PVL prophage; 77likevirus, PVL groups 1 (ϕSa2wa-st22) and 3 (ϕSa2wa-st59) lysogenised ST22-IVc and ST59-V respectively and 3alikevirus, PVL group 2 prophage (ϕ2958 and ϕSa2wa-st1) lysogenised ST5-II and ST1-IVa respectively.

Alpha alignment of colinear regions of ϕSa2wa-st1, -st5, -st59 and -st93 identified novel modules and heterogenous genes

The DNA processing main module is the most variable region in PVL bacteriophages and Fig 4 presents Alpha alignments of colinear sections of ϕSa2wa-st1, -st5, -st59 and -st93 from the 5' end of the bacteriophages. The region encodes conserved genes associated with lysogeny, and a variable region of early transcribed genes associated with lysogeny, bacteriophage defence and regulation. Variable genes and different colinear modules with similar functions can be detected in this graphical representation of heterogeneity.

Fig 4

A and B. Alpha alignment of colinear sections of ϕSa2wa-st1, ϕSa2wa-st5, ϕSa2wa-st59 and ϕSa2wa-st93. Each genome in the alignment is assigned an uppercase letter. Alignment positions for the corresponding genome are indicated in parentheses alongside the letter. Anchor sequences are similar segments of significant length shared by all genomes in the alignment. Nodes are gapless alignments specific for individual genomes in the alignment; they display the length and percent identity of the aligned region; unless otherwise indicated identity is 100%. Anchors and nodes are connected by color-coded arrows, one color for each genome and numbered sequentially on the figure. Dotted arrows replace nodes of less than 20 bp. Abbreviations: C, ϕSa2wa-st1; D, ϕSa2wa-st5; K, ϕSa2wa-st59; O, ϕSa2wa-st93; WA, Western Australian; INT, International.

Fig 4A has 15 alignment nodes. The anchor sequences are nodes 1 and 14 which encode int with the 5' end of orfC (exemplified by ϕSa2wa-st5, Genbank: AUM57679 and AUM57680) and the 5' terminal of a helix-turn-helix (HTH) domain-encoding ORF (Genbank: AUM57693) respectively (Fig 1). The genomes diverge at bp 1,913 (Alpha alignment node 1). Following two or three HPs of unknown function ϕSa2wa-st1 (c3081-3542), -st5 (c2946-3416) and -st93 (c3080-3550) encode a putative toxin gene, (Genbank: AUM57684, 72–89% nt identity, 82.8–94.9% aa similarity) (Fig 1). The ϕSa2wa-st1 and -st93 putative toxin sequences (77.3% nt identity) diverge at bp 3,308 and 3,307 respectively (Alpha alignment node 5). All polymorphisms in these ORFs are in the 5' ends and most (27/38) are non-synonymous. Non-random distribution of polymorphisms such as this indicates homologous recombination between divergent genes has probably occurred. ϕSa2wa-st5 has a gas vesicle protein G (GvpG) domain-encoding ORF (Genbank: AUM57683) downstream of the toxin gene (Genbank: AUM57684, Alpha alignment node 7) which has 89% nt identity with the ϕSa2wa-st1 toxin gene. The ϕSa2wa-st1 and -st5 toxin genes are followed by a xenobiotic response element (XRE)-HTH putative transcriptional regulator ORF which also encodes a MqsA antitoxin superfamily domain (Genbank: AUM57685, Alpha alignment node 8, 99.4% nt identity), while ϕSa2wa-st93 encodes a XRE-HTH transcriptional regulator (Genbank: AUM58233, Alpha alignment node 7). ϕSa2wa-st1 and -st5 then encode a XRE_HTH family regulator followed by a bacteriophage pRha superfamily domain-containing protein that interferes with infection of strains that lack integration host factor (Genbank: AUM57686 and AUM57687, Alpha alignment node 8).

When compared with all the WA-PVL prophages the node 8 module of ϕSa2wa-st1 and -st5 is shared with only ϕSa2wa-st30, and the node 7 module of ϕSa2wa-st93 is shared with only the ϕSa2USA/ϕSa2wa-st93 group. ϕSa2wa-st59 encodes a unique 4,658 bp section (bp 2511–7168), which encodes two XRE family transcriptional regulators, a bacteriophage anti-repressor and 10 putative HPs (AUM57878 to AUM57891, Alpha alignment node 8). Nodes 13, 14 and 15 encode the 5' end of a HP ORF (Genbank: AUM58237) with a common core (Alpha alignment node 14) and divergence by ϕSa2wa-st5 and ϕSa2wa-st1 indicated by the graph.

Following two to four heterogenous colinear HP ORFs the Fig 4B Alpha alignment node 27 reveals two unrelated colinear modules. ϕSa2wa-st5 and -st59 encode ORFs for a bacteriophage Mu Gam-like protein which protects double stranded DNA from exonuclease degradation, two overlapping single-stranded binding proteins and a putative HNHc_6 superfamily nuclease (Genbank: AUM57696 to AUM57698) which are not shared by any other WA PVL prophage (Fig 1). ϕSa2wa-st1 and -st93 encode three overlapping ORFs encoding a HP, a Cas4-like protein and a DUF2185 protein (Genbank: AUM58242, AUM58243 and AUM58244). This module may be a defence system or part thereof against the bacterial CRISPR-Cas system and it is shared by all study prophage (97–100% nt identity) except ϕSa2wa-st5, -st22, -st59 and -st772. ϕSa2wa-st1 and -st93 then encode DNA polymerase A (Alpha alignment node 28) while ϕSa2wa-st5 (Fig 1) and -st59 encode DNA polymerase C.

Alpha aligner-defined nodes reveal extensive mosaicism and recombination in PVL bacteriophage from WA

The singleton ST93 genome is stable and well-adapted in the geographical region. To further investigate the mosaicism in the prophages Table 3 presents the nodes of ϕSa2wa-st93 as determined by the Alpha aligner in Fig 4A and 4B, identifies the putative proteins or protein sections and shows the local prophages that encode the same sequence with 97 to 100% nt identity. ϕSa2wa-st8, -st93mssa and ϕSa2USA are almost identical to ϕSa2wa-st93 and have been excluded from the Table. There is evidence of extensive recombination. The only prophage that was identical in this region was the closely related ϕSa2wa-st72 and the only prophage not to share any module was ϕSa2wa-st772. Most of the shared sequence involved the prolate-headed 3alikevirus prophages however, there was also evidence of recombination with the 77likevirus icosahedral-headed ϕSa2wa-st59. The Alpha aligner defined modules consist of split genes, single genes, groups of genes and intergenic regions, some shared by several prophages and others by only one or two. At 96.6% nt identity ϕSa2wa-st72 is a member of the ϕSa2USA/ϕSa2wa-st93 group in this study and the Table 3 modules with homology only with ϕSa2wa-st72 represent modules encoding functions that, amongst the prophages in this study, are unique to this successful bacteriophage.

Table 3

Alpha aligner defined nodes of ϕSa2wa-st93 and node-associated ORFs or intergenic regions having 97–100% sequence identity with other WA PVL prophages.

ϕSa2wa-st93 Position/Node*	Protein(s) accession no’s, HPs or regions	WA PVL bacteriophages with 97–100% sequence identity
22-1913/1	Integrase, AUM58227; Split HP, AUM58228	ϕSa2wa-st1, -st5, -st22, -st30, -st59, -st72, -st78, -st80 -st121mssa
1914-2140/2	Split HP, AUM58228; split HP AUM58229	ϕSa2wa-st5, -st72, -st78
2146-3307/5	Split HP, AUM58229; HP, AUM58230; HP, AUM58231; split Toxin, AUM58232	ϕSa2wa-st1, -st72
3308-4724/7	Split Toxin, AUM58232; HP, AUM58233; HP, AUM58234; HP, AUM58235	ϕSa2wa-st72
4725-4766/11	Intergenic region	ϕSa2wa-st1, -st5, -st30, -st59, -st72, -st78, -st80, -st121mssa
4767-5017/12	HP, AUM58236	ϕSa2wa-st72
5018-5060/13	Split HP, AUM58237	ϕSa2wa-st1, -st30, -st59, -st72, -st78
5061-5096/14	Split HP, AUM58237	ϕSa2wa-st1, -st5, -st30, -st59, -st72, -st78, -st80, ϕ -st121mssa
5097-5163/15	Split HP, AUM58237	ϕSa2wa-st5, -st30, -st59, -st72, -st78, -st80, -st121mssa
5341-5423/18	Split DUF1270, AUM58238	ϕSa2wa-st72, -st78
5424-5446/19	Split DUF1270, AUM58238	ϕSa2wa-st72, -st78
5472-5497/22	Intergenic region	ϕSa2wa-st72
5498-5828/23	HP, AUM58239; split DUF2482 HP, AUM58240	ϕSa2wa-st59, -st72
5829-6178/24	Split DUF2482 HP, AUM58240; split DUF1108 HP, AUM58241	ϕSa2wa-st72
6179-6361/25	Split DUF1108 HP, AUM58241	ϕSa2wa-st72
6362-6422/26	Split DUF1108 HP, AUM58241	ϕSa2wa-st5, -st59, -st72
6429-8560/27	HP, AUM58242; Cas4-like, AUM58243; DUF2815 HP, AUM58244	ϕSa2wa-st1, ϕ -st30, -st72, -st78, -st80, -st121mssa
8565-10659/28	DNA polymerase A, AUM58245; split DUF3113 HP, AUM58246	Sa2wa-st1, -st30, -st72, -st78, -st80, -st121mssa

Proteins and hypothetical proteins are indicated by their Genbank protein-id number. Genbank domains of unknown function are indicated; Split proteins represent split open reading frames. Abbreviations: DUF, domain of unknown function; HP, hypothetical protein; no’s, numbers

*As presented in Fig 4

With between 77% and 82.9% nt identity ϕSa2wa-st5 from WA3 was the most distantly related of the 3alikevirus group and was successfully induced and therefore probably transmissible (Table 4). It had highest homology with ϕSa2958 (Genbank: AP009363; 99% nt identity over 72% of the genome) however, this was essentially in the structural morphology and lysis, virulence and lysogeny modules. Fig 1 presents a diagrammatic comparison of ϕSa2wa-st5, -st93 and -st1 with the ϕSa2wa-st5 ORFs coloured according to the WA PVL-prophages that shared 97–100% sequence identity. With the exception of the bp 15,838 to 17,040 module which was homologous with non-PVL bacteriophage 53 (Genbank: AY954952; 100% nt identity) the unshared regions of ϕSa2wa-st5 encoding multiple ORFs were unique. As well as random mutations that occur during chromosomal replication this heterogeneity indicates that horizontal recombination has occurred between the bacteriophage during their evolution.

Table 4

Bacteriophage induction and lysogenisation of historic PVL-negative WA CA-MRSA.

Lysogen	Lysogenised recipientsTotal pfu/mL		PVL positive plaques	Induced PVL bacteriophage	PVL-negative CA-MRSA lysogenised
	RN4220	WBG286
MW2	>1x105	0	>100	ϕSa2mw	WA5WBG7583
WA115798	0	0	0	0	NA
WA2RNSH95	0	1x103	0	0	NA
WA318790	2x103	0	20	ϕSa2wa-st5	None
W17S	0	1x102	1	ϕSa2wa-st93mssa	Not tested
K25S	3x102	0	3	ϕSa2wa-st121mssa	Not tested
Qld Clone16790	0	1x102	1	ϕSa2wa-st93	WA5WBG7583

Abbreviations: NA, not applicable; pfu, plaque forming units.

in-vitro induction of PVL-positive ϕSa2 from Australian S. aureus and lysogenisation of historic PVL-negative WA CA-MRSA

To test the transmissibility of PVL-positive ϕSa2 lysogenising the Australian S. aureus and the lysogenic capabilities of the historic PVL-negative WA CA-MRSA (Table 1), in-vitro induction, propagation and lysogenisation experiments were performed (Table 4).

Bacteriophage were induced from all isolates tested except WA115798. Overall, only two of the five indicator strains, RN4220 and WBG286 were lysogenised and specifically, only one in each induction experiment, demonstrating some specificity of lysogenisation (Table 4). Hybridisation of the plaques revealed that ϕSa2wa-st5, -st93mssa, -st93, -st121mssa and the control, ϕSa2mw, were induced out of their lysogens. ϕSa2wa-st78 may not have been induced because it lacked an evident attLi integration site (Fig 1) however, the reason why ϕSa2wa-st1 was not induced is currently unclear.

ϕSa2wa-st121mssa could not be propagated to a sufficiently high titre in-vitro however, ϕSa2wa-st5, -st93 and the control, ϕSa2mw, were tested for their ability to lysogenise all of the historic PVL-negative WA CA-MRSA. WA5WBG7583 was lysogenised with ϕSa2wa-st93 and the control, ϕSa2mw, but not ϕSa2wa-st5. None of the other PVL-negative WA CA-MRSA were lysogenised in-vitro with any of the induced and propagated bacteriophages.

Discussion

The genomes of PVL prophages from temporally and geographically related S. aureus of local and international origin have revealed an unexpected amount of diversity that has made it difficult to trace their origins. There has been recombination between bacteriophage of the same and different genera as well as genetic diversity in the chromosomal integration sites, the regulation regions, the defence, DNA-processing, structural and packaging modules and the lukSF-PV genes. There was no evidence that the icosahedral-headed prophages from international clones of CA-MRSA had transferred to the WA clones. The prolate-headed prophage formed the largest group however, with the exception of ϕSa2wa-st8, they were so diverse it was not possible to determine if there had been horizontal transmission of whole bacteriophages. There has been recombination between the international and local prolate-headed bacteriophage and, to a lesser extent, also between prolate- and icosahedral-headed bacteriophage that are present in WA at some stage during their evolution.

With 99.97% sequence identity, it is evident that WA518751 has probably acquired ϕSa2wa-st8 in the WA community from either a ST93 S. aureus or USA300. ϕSa2wa-st93 was induced in-vitro and then it lysogenised PVL-negative WA5WBG7583 demonstrating that this clone can accept the bacteriophage. On-the-other-hand, WA518751 and USA300 had identical ϕSa2 integration-site sequences indicating that the bacteriophage could also have been horizontally transmitted from USA300.

ϕSa2wa-st8, -st72, -st93, -st93mssa and ϕSa2USA probably represent a single bacteriophage that has transmitted between lineages of S. aureus. USA300 and the Queensland clone are two of the most virulent and widely disseminated CA-MRSA and in this and a previous study [46] it is evident that there has been horizontal transmission of a ϕSa2USA/ϕSa2wa-st93-type bacteriophage between their CC8 and Singleton 93 ancestors, however there is no indication of when or where this occurred. USA300 acquired ϕSa2USA in North America following importation of its ancestor in the early 20th century [47]. ϕSa2wa-st93mssa was present in ST93-MSSA, the most prevalent colonizer in remote WA in 1995, and this clone was the ancestor of the Queensland clone that emerged in Queensland, Australia in the early 2000’s [14, 48, 49]. ϕSa2wa-st93mssa was well adapted in ST93-MSSA and Australia before the clone acquired the SCCmec and before USA300 was imported into Australia [22]. Against the background of PVL prophage diversity revealed in this study it is extraordinary that the ϕSa2USA/ϕSa2wa-st93 bacteriophage has remained stable over at least 20 years in different geographic and genetic environments. To gain insights into the success of this bacteriophage it would be informative to investigate the putative proteins of unknown function encoded by the unique modules of ϕSa2wa-st93 revealed in Table 3.

The international Korean CA-MRSA clone is characteristically PVL-negative [50] and has probably acquired ϕSa2wa-st72 in the WA community. This may represent a recent acquisition of a ϕSa2USA/ϕSa2wa-st93 bacteriophage with the prophage undergoing gradual changes as it adapts to a CC72 background and different geographical conditions. As with the WA PVL-positive CA-MRSA the PVL-positive Korean clone has not thrived and forms only 0.02% of CA-MRSA in the WA community [19].

The diversity in shared genes such as the hypothetical proteins that have been split in Table 3 according to their homologies with all the prophage in the study is interesting. In the putative toxin genes, all of the nucleotide differences were in the 5' end of the ORF and most resulted in different amino acids. This may be a defence strategy that has involved recombination within genes resulting in proteins with the same function but different antigenic profiles.

With the exception of two pairs of prophage all had distinct integration-site sequences however, the impact of this on the specificity of lysogenisation is currently unknown. The effect of lysogeny by ϕSa2 on host fitness could not be determined. The preferred insertion site was within a lipoprotein_7 domain-encoding ORF within a paralogous cluster of three or four ORFs that encoded a DUF1672 lipoprotein. As has been previously reported, the ORFs truncated by ϕSa2wa-st22, -st30 and -st80 type bacteriophage [37] and now ϕSa2wa-st121, were different, however they were similarly positioned within the same DUF1672 lipoprotein cluster. Lipoproteins serve as transporters of nutrients and contribute to virulence and fitness in S. aureus and increased complements have been associated with particularly pathogenic strains [38]. The impact of truncation of the ϕSa2 target genes on host fitness requires further investigation.

Prophage lysogenising 11 lineages of S. aureus have been investigated in this study and adaptation to different genetic backgrounds is undoubtedly one of the reasons for the diversity observed. Investigation of more genomes of PVL prophage from S. aureus belonging to the same genetic lineage is now required. The low occurrence of PVL-positive variants of established PVL-negative CA-MRSA in the WA community suggests that the clones may not have adapted well to the acquisition of PVL-positive ϕSa2.

17 Sep 2019

PONE-D-19-19758

Diversity of Bacteriophages Encoding Panton-Valentine Leukocidin in Temporally and Geographically Related Staphylococcus aureus

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Reviewer #1: This is a highly detailed genomic analysis of recombinational interactions among PVL-encoding prophages in Western Australia (WA), including data indicating that recently imported S. aureus strains with PVL-encoding prophages contributed to the remarkable recent increase in PVL prevalence in WA. Given that the phenomenon of recombinational promiscuity among phages is very well known and has been thoroughly documented years ago, this paper represents merely a catalog of this phenomenon as applied to a particular set of prophages. Although a useful addition to the general literature on recombinational promiscuity among prophages, it does not present any novel principles or ideas. Moreover, the authors employ an elaborate prophage and strain nomenclature, which makes the presentation highly confusing, nearly impenetrable. It would help greatly if this were radically simplified. Finally, although there is a hint that the paper is about the impact of foreign strains on the prevalence and spread of PVL among prophagres in WA, it is never clearly stated as the story behind this study. If this is, in fact, the motivation, it should be clearly stated up front and detailed in the discussion.

A couple of minor points:

1. Please explain how these strains were introduced into Australia.

2. It is implied, line 236, that��Sa2wa-st78 used a different att site, but the data in Fig. 1 do not support this – please clarify.

3. Were any matching sequences between otherwise unrelated genes encountered, that could account for recombination, other than that mentioned in line 276?

4. Could the authors show (tabulate) which imported PVL phages lysogenized native strains intact, and which segments of these were incorporated by recombination into native prophages?

Reviewer #2: Overview: The paper by O’Brien et al. investigates the diversity of prophages carrying PVL in different isolates of CA-MRSA from Western Australia. Before 2005, all CA-MRSA isolates from this region were PVL negative. This paper seeks to explore the genetic diversity of PVL encoding prophages from WA to try and identify their origin. The authors hypothesize that the PVL phage may have been introduced to WA strains via horizontal transmission from international PVL positive CA-MRSA strains.

The paper is excellently written and investigates, in great depth, the similarities and differences between the bacteriophages in question. There are no major concerns with the work (mostly bioinformatic) outlined in the manuscript, although some improvements could be made to improve the readability of the manuscript for non-experts in the field (comments below).

Major comments

• The major concern with this manuscript arises from the questionable impact/result of the study. The results of the study are summed up by the authors themselves in the discussion as: “The genomes of PVL bacteriophages … have revealed an unexpected amount of diversity that has made it difficult to trace their origins.” Essentially this is the concern. An in-depth analysis of 14 bacteriophages ultimately revealed there was too much diversity to make any solid conclusions regarding their origin.

• More detail. There are numerous places throughout the manuscript where insufficient information is given which makes the data difficult to read/interpret. Examples include;

o Results section “Sequence analysis”. The prophages were sorted into different genera depending on “morphogenesis and structural genes.” What were these criteria?

o There are genera in this section that are mentioned but not fully described, i.e, the “3alikevirus” genus. To me, this doesn’t mean anything without a further definition of this genus. This may be common knowledge among phage biologists but not to others.

o Titles for the results section are uninformative. Titles like “sequence analysis, PVL gene sequencing, alpha alignments etc.” do not provide readers with any information on conclusions drawn.

o The results section “Induction and lysogenisation” (and corresponding Table 4) is confusing and lacking in detail. The authors should clearly state what was done, (i.e what strains were induced), which ones they were able to obtain bacteriophage from, why they used those strains, which recipient strains they attempted to infect …etc. Several columns in Table 4 are unexplained (e.g. lukSF-PV – pos plaques. This is the only experimental section in the manuscript and it is unclear and difficult to interpret.

• Data. The authors do not indicate if the bacteriophage sequences generated have been deposited in an online repository.

Minor criticisms:

• The designation for strains/prophages used in the study is confusing and limits readability for those not familiar with this type of nomenclature. The titles are very lengthy and they make it difficult to keep track of which isolate is being discussed. While I acknowledge it may be difficult if they could be simplified it would make the paper much clearer.

• Authors alternate the spelling of “defense” and “defence” throughout the manuscript. Please adjust to make uniform.

• In vitro is not always italicized, one example is in line 421.

• Figure 4 is difficult to interpret. The colors on the key don’t always match the colors on the actual figure. Specifically, the purple color designating ϕSa2wa-st80 either doesn’t appear on the figure or is a totally different shade on the figure. The figure legend could be expanded to help explain the color code. It would also be useful to indicate the areas shown in Fig 1 and 2.

• Line 187: “Fourteen prophages…(Table 1)”. Why does Table 1 have 17 phages listed?

• Line 278: (Figure 4). Figures are out of order

• Line 262 to 275. Overall the writing in this section was unclear and I was unable to follow the impact of Sa2wa-st772

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25 Oct 2019

We thank the reviewers for their time spent reviewing the manuscript and appreciate their suggestions. The author agrees that some parts of the presentation are confusing and difficult to read and acknowledges both reviewers for requesting simplification of nomenclature. The following changes have been made:

• In the Materials and Methods section the bacteriophage/prophage nomenclature has been explained and the author respectfully requests permission to leave it unchanged. So far there has not been any standardization of nomenclature for PVL bacteriophage in S. aureus. Following discussions with colleagues, the authors felt it was important for this and future publications to apply a meaningful nomenclature that would show at a glance which bacteriophage/prophage they were, their geographical provenance and the genetic background of their lysogen. To make the manuscript easier to read, where the bacteriophage occur in groups in the same sentence full designation has been given to only the first bacteriophage, for the remainder the prefix in front of the hyphen has been removed and only the bacteriophage -st designations separated by commas have been presented. Furthermore, to reduce the number of prophages mentioned, where the Sa2USA/Sa2wa-st93 group of bacteriophages had the same properties, the individual bacteriophages have been removed and the group designation inserted.

• Clone designations have been shortened by presenting the strain identity number as a subscript.

• Clone genotypes have been shortened by removing “MRSA” and the bracketed SCCmec descriptors from the designations.

• In addition to the changes requested by the reviewers the author feels that the second paragraph in the PVL gene sequencing section was overly complicated and difficult to comprehend. For several years now scientists have been sequencing the PVL genes and most publications have associated different alleles with the genetic lineage of the lysogen, however, this study shows that this is not the case, therefore we feel compelled to report the findings in detail. No information has changed. The main points have been numbered and sentences have been re-arranged to make the paragraph more comprehensible.

Reviewer #1

1. This is a highly detailed genomic analysis of recombinational interactions among PVL-encoding prophages in Western Australia (WA), including data indicating that recently imported S. aureus strains with PVL-encoding prophages contributed to the remarkable recent increase in PVL prevalence in WA.

The study was performed due to concern that the PVL bacteriophages from international MRSA clones were moving into the predominant local clones. We did not mean to imply that the international clones were responsible for the high rate of PVL-positive MRSA in WA. To address this issue, we have modified the text as follows:

• In the Abstract, a sentence has been added. “There was concern that PVL bacteriophages from the international clones were transferring into the local clones, therefore a comparative study of PVL-carrying Sa2 prophage genomes from historic WA PVL-positive S. aureus and representatives of all PVL-positive CA-MRSA isolated in WA between 2005 and 2008 was performed.”

• In the Introduction: Sentence change. “The rise in the number of PVL-positive CA-MRSA in WA since the first was found in 2003 has been alarming.”

2. Given that the phenomenon of recombinational promiscuity among phages is very well known and has been thoroughly documented years ago, this paper represents merely a catalog of this phenomenon as applied to a particular set of prophages.

• What this study is trying to do is further the scientific understanding of bacteriophage recombination and heterogeneity by using the Alpha aligner to identify modules and investigate at the sequence level exactly where the greatest diversity is, what form it takes and which modules are being exchanged.

• We acknowledge the known high rate of recombination by bacteriophages and in the Introduction extra information has been added as follows:

“It has been well documented that bacteriophages undergo high rates of recombination and both these forms of transmission allow opportunity for genetic exchange, the potential mechanisms being transposition, site-specific recombination, homing endonucleases and homologous and illegitimate recombination (11).”

Minor points

1. Please explain how these strains were introduced into Australia.

• In the Introduction: New sentence. “In WA, all MRSA are submitted to a central facility for typing and epidemiological investigation (21) and between 2005 and 2008 eight international PVL-positive CA-MRSA were introduced into WA.”

2. It is implied, line 236, thatSa2wa-st78 used a different att site, but the data in Fig. 1 do not support this – please clarify.

• There is no attLi for Sa2wa-st78 in Fig 2. It could not be identified.

• We have commented on this in the following unchanged sentence:

“ attLi of Sa2wa-st78 could not be identified, however its attRi was reasonably similar (3 bp difference) to the Sa2wa prophages over the LH arm and the first 17 bp of the common core while 32 of the remaining 37 bp were different (Fig 2)”

• In the last paragraph of this section the difference is further discussed.

3. Were any matching sequences between otherwise unrelated genes encountered, that could account for recombination, other than that mentioned in line 276?

• There were matching sequences throughout however, given the plethora of mechanisms of recombination employed by bacteriophages we would not dare to speculate on how any recombinations may have occurred unless we could show evidence of the event (such as flanking repeats).

4. Could the authors show (tabulate) which imported PVL phages lysogenized native strains intact, and which segments of these were incorporated by recombination into native prophages?

• A Table would be too small. Only one PVL-positive native WA clone had a bacteriophage that was identical (97-100% homology) to one of the imported bacteriophages. That was WA518751 lysogenised with Sa2wa-st8 which has been incorporated into the Sa2USA/Sa2wa-st93 group.

• Given the heterogeneity of the bacteriophages, the vastness of the geographical region and community they came from, and how very little is understood about bacteriophage recombination it would not be possible from this study to reliably predict where individual segments originated.

Reviewer #2

Major comments

1. The major concern with this manuscript arises from the questionable impact/result of the study. The results of the study are summed up by the authors themselves in the discussion as: “The genomes of PVL bacteriophages … have revealed an unexpected amount of diversity that has made it difficult to trace their origins.” Essentially this is the concern. An in-depth analysis of 14 bacteriophages ultimately revealed there was too much diversity to make any solid conclusions regarding their origin.

• With the exception of the Sa2USA/Sa2wa-st93 group the authors were unable to get any bootstrap support for any phylogenetic analysis on this collection of PVL bacteriophage. This is remarkable in itself. The authors feel that the PVL prophage may be providing the lysogens with fitness properties other enhanced virulence from PVL production. This is why this study has looked so closely at the chromosomal attachment region and searched for novel or unique bacteriophage genes and modules. This will be for future studies.

2. The prophages were sorted into different genera depending on “morphogenesis and structural genes.” What were these criteria? There are genera in this section that are mentioned but not fully described, i.e, the “3alikevirus” genus. To me, this doesn’t mean anything without a further definition of this genus.

• The most recent reference on staphylococcal bacteriophage classification has been used. The classifications are based on the criteria therein. To direct readers to the references more clearly we have re-written a sentence as follows:

“The prophage genomes had the organisation of Siphoviridae family Sfi21-like PVL viruses of the Caudovirales order and, according to the most recent staphylococcal bacteriophage classification criteria, were placed into two genera and three PVL bacteriophage groups (Table 2) (34-36).”

• The most striking difference between the genera is that the 77likevirus genus consists of icosahedral-headed bacteriophage and the 3alikevirus genus are prolate-headed bacteriophage. This has been mentioned in the text and reinforced throughout the document.

3. Titles like “sequence analysis, PVL gene sequencing, alpha alignments etc.” do not provide readers with any information on conclusions drawn.

• The Results headings have been changed as requested.

4. The results section “Induction and lysogenisation” (and corresponding Table 4) is confusing and lacking in detail. The authors should clearly state what was done, (i.e what strains were induced), which ones they were able to obtain bacteriophage from, why they used those strains, which recipient strains they attempted to infect …etc.

• This section has been re-written to address the reviewers’ concerns. To keep consistency with the main focus of the study only induction experiments on the Australian clones have been reported and more details on what was done have been included. The author agrees that presenting the results of the international clones only added confusion. The Table has been re-formatted to include only the Australian clones with the control, and an extra column has been added to present the results of the different recipient/indicator strains.

• One sentence giving extra information has been added:

“The induced bacteriophages each lysogenised only one of the two indicator strains demonstrating some specificity of lysogenisation.”

5. Data. The authors do not indicate if the bacteriophage sequences generated have been deposited in an online repository.

• In the original submission, this data was under the heading “Sequence data availability” at the end of the Results section. It has now been moved to the first paragraph of the Results section to make it more prominent.

Minor criticisms

1. Authors alternate the spelling of “defense” and “defence” throughout the manuscript. Please adjust to make uniform.

• This has been done.

2. In vitro is not always italicized, one example is in line 421.

• This has been done.

3. Figure 4 is difficult to interpret. The colors on the key don’t always match the colors on the actual figure. Specifically, the purple color designating ϕSa2wa-st80 either doesn’t appear on the figure or is a totally different shade on the figure.

• The colors have been adjusted.

4. The figure legend could be expanded to help explain the color code.

• The following sentence has been adjusted in the Figure 4 legend:

“Sa2wa-st5 ORFs are represented as arrows indicating the direction of transcription and coloured according to the PVL prophage or groups of PVL prophages from PVL-positive S. aureus in WA that share 97 to 100% nucleotide identity.”

5. It would also be useful to indicate the areas shown in Fig 1 and 2.

• The Figure 1 (now Fig 2) sequences are attachment sites that are on the chromosome, proximal to the terminals of the prophages. This is explained in the text. “Chromosomal sequences proximal to the prophage terminals encoded the hybrid attLi and attRi sites of the attB and attP sites on the chromosome and a circularly permuted form of the bacteriophage.” Because the prophage sequences were extracted from whole genomes it was not possible to determine which hybrid att site belonged to the bacteriophage and which was on the chromosome.

• The Figure 2 (Now Fig 3) region is in a part of Figure 1 that already is “cluttered” with information. The relevant ORFs are mentioned in the text and marked on Fig 1. To guide readers to the region in the text we have inserted a reference to Fig 1 as follows:

“The intergenic region between the divergently transcribed integrase gene, int, and its associated HP ORF, originally called orfC (39) (Fig 1), contained structures indicative of involvement in regulation and lysogeny in all prophages (Fig 3).”

6. Fourteen prophages…(Table 1)”. Why does Table 1 have 17 phages listed?

• Table 1 in the manuscript submitted has 14 prophages listed. The resubmitted manuscript has Table 1 with 14 prophages.

7. Line 278: (Figure 4). Figures are out of order

• The Figures have been re-numbered and re-inserted in the correct order

8. Line 262 to 275. Overall the writing in this section was unclear and I was unable to follow the impact of Sa2wa-st772

• This is the first description of the secondary genetic features which are predicted to be involved in lysogeny and regulation in Sa2 bacteriophages. The authors respectfully feel that the text, although somewhat complicated is supported by the figure and provides a guide for further investigations.

• The impact of the differences in Sa2wa-st772 have been addressed in a new paragraph in this section.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Dec 2019

PONE-D-19-19758R1

Diversity of Bacteriophages Encoding Panton-Valentine Leukocidin in Temporally and Geographically Related Staphylococcus aureus

PLOS ONE

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Reviewer #2: My major concern with the previous version of the manuscript has not been addressed. While the authors present an extremely detailed analysis of 14 bacteriophage genomes there doesn't appear to be any solid conclusion or impact from the study. The manuscript has been modified to make it clearer and easier to understand but the overall impact of the study remains unclear.

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20 Jan 2020

Reviewer #2: My major concern with the previous version of the manuscript has not been addressed. While the authors present an extremely detailed analysis of 14 bacteriophage genomes there doesn't appear to be any solid conclusion or impact from the study. The manuscript has been modified to make it clearer and easier to understand but the overall impact of the study remains unclear.

PVL-positive CA-MRSA have evolved to become one of the most significant bacterial pathogens in global communities. From an initial concern that well adapted local clones of PVL-negative CA-MRSA were acquiring the PVL-carrying bacteriophage from imported international CA-MRSA and consequently, the implications for morbidity and therapeutic management of infections in WA, the authors have told a story of the PVL-carrying bacteriophage in a defined geographical region from a point in time when there were no PVL-positive CA-MRSA to a situation where 52.8% of all CA-MRSA in WA were PVL positive. So far this is the only study that has addressed the epidemiology and genetics of a temporally and geographically related virulence-carrying staphylococcal bacteriophage.

Heterogeneity amongst bacteriophages is acknowledged and for the majority of bacteriophages in this study this is what was ultimately revealed however, one bacteriophage was shown to be remarkably stable over at least 20 years in different genetic backgrounds. In a time of the emergence of multiple antibiotic resistance in S. aureus, when several groups are investigating the utility of bacteriophages as therapeutic agents, this finding is important. If bacteriophages are to be used as therapeutic agents then their epidemiology and basic biology, such as regulation of lysogeny, need to be understood. Why is this bacteriophage so stable and successful in particularly virulent CA-MRSA? This study has used the Alpha Aligner to identify unique genes that may help answer this question. Furthermore, use of the Alpha Aligner has added to the understanding of basic bacteriophage biology by enabling a detailed analysis of the genetics of the most diverse region of the bacteriophages.

There is a paucity of information on the molecular biology of staphylococcal bacteriophages and the author respectively feels that the overall impact of the manuscript is that it that it presents new knowledge that can be used for further investigations into bacteriophage biology and genetics in CA-MRSA.

Changes to the manuscript

• The Abstract has been modified. The following statement has been removed because the authors did not feel it was relevant to the abstract:

“…however the role played by the toxin and the bacteriophage in S. aureus virulence has been controversial.”

• In the abstract the clones that probably acquired the PVL bacteriophage in the WA community have been named and reference to the fact that they did not disseminate has been included.

• To enable clearer assessment of the impact of the manuscript the focus of the study has been made clearer in the Introduction by numbering and a more logical reversing of the two aims. This is followed through in the Abstract.

• Throughout the manuscript reference to the chromosomal attachment site as the “attachment site” has been changed to “integration site”. This is to avoid confusion with the cell wall bacteriophage attachment site that is used in many publications.

• A comma has been deleted and another inserted to improve grammar and readability of the following sentence. “Overall, only two of the five indicator strains, RN4220 and WBG286 were lysogenised and specifically, only one in each induction experiment, demonstrating some specificity of lysogenisation (Table 4).”

• In the Discussion. In the first sentence the word “prophages” has replaced “bacteriophages”.

• In reference 44. The Journal has been correctly abbreviated.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

22 Jan 2020

Diversity of Bacteriophages Encoding Panton-Valentine Leukocidin in Temporally and Geographically Related Staphylococcus aureus

PONE-D-19-19758R2

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29 Jan 2020

PONE-D-19-19758R2

Diversity of Bacteriophages Encoding Panton-Valentine Leukocidin in Temporally and Geographically Related Staphylococcus aureus

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