Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire
© Votintseva et al.; licensee BioMed Central Ltd. 2014
Received: 11 December 2013
Accepted: 4 March 2014
Published: 12 March 2014
Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as “spa-types”, based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as “non-typeable”.
We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated “non-typeable” using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies.
This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.
Staphylococcus aureus is a commensal organism that colonizes nasal mucosa in 25-30% of the healthy human population [1–6] and is responsible for a wide range of human diseases including serious nosocomial infections. S. aureus encodes many virulence factors including the surface Ig-binding protein A (spa) whose function is to capture IgG molecules in the inverted orientation and therefore prevent phagocytosis of the bacterial cells by the host immune system [7–12].
Typing the highly variable Xr region of the spa-gene is one of the most common methods for genotyping S. aureus. Even if well-established genotyping methods like MLST are indispensable, spa-typing has major advantages due to its high discriminatory power, typing accuracy, speed, reproducibility and ease of interpretation. Spa-typing also facilitates communication and data comparison between national and international clinical laboratories . However, one weakness of current spa-typing methods is that rearrangements in the in the IgG-binding region of the gene, where the forward spa-primer is located, lead to 1-2% of strains being designated “non-typeable”. Five non-spa-typeable S. aureus clinical strains with rearrangements in the IgG-binding domain of the spa-gene were first described by Baum et al. in 2009 . Although artificially constructed spa-deficient S. aureus strains are used in laboratory experiments [15–18], only a few other studies have reported variants isolated from human and cattle with rearrangements in the spa-gene [19–24]. Missing particular variants that cannot be typed may affect inferences about genotype associations. Whilst the prevalence of such rearrangements can be directly estimated from the proportion of non-typeable strains, detecting rearrangements that do not affect spa-typing would require sequencing the whole spa-gene; nevertheless such rearrangements may still be informative with respect to population structure.
Further complexity is introduced by the fact that most studies type only one colony per sample, thus assuming S. aureus colonization is by a single strain and likely systematically underestimating the number of spa-types per individual. The presence of non-typeable S. aureus strains with rearrangements in the spa-gene increases the number of undetected circulating spa-types even further.
Here we therefore developed a new set of primers to amplify the spa-gene from all formerly non-typeable S. aureus samples regardless of the specific spa-gene rearrangement. We used our modified spa-typing protocol to investigate the nature and proportion of strains with rearrangements in the S. aureus spa-gene in two large studies of community nasal carriers and inpatients, and the potential impact of S. aureus protein A mutants on epidemiological studies.
Collection of isolates
Nasal swab samples were collected from consenting individuals recruited from five General Practices (GP) in Oxfordshire that were members of the Thames Valley Primary Care Research Partnership, National Institute of Health Research (approved by Oxfordshire Research Ethics Committee B, reference 08/H0605/102, ). On recruitment, a nasal swab was taken from each individual by a research nurse. Participants were trained in self-swabbing and all participants who were culture-positive for S. aureus (n = 360) on recruitment and one quarter (n = 211) of initially culture-negative participants were sent a self-swabbing kit after one month and then every two months. Swabs in charcoal medium were returned by mail and stored at 4°C before processing. During the 36 months of the study, S. aureus was isolated from at least one swab of 442 individuals yielding 3905 samples which were spa-typed and analyzed here.
S. aureus isolates were obtained from samples collected from the Intensive care Unit (ITU), Gerontology and Trauma wards of the John Radcliffe hospital in Oxford as a part of routine screening for inpatients for infection control surveillance. For all three wards, nasal swabs were collected from individuals at ward admission and discharge as well as once a week during their stay within the ward . All swabs were taken by nurses, as described above, and were processed by the routine laboratory at the John Radcliffe hospital, Oxford. In total, S. aureus was isolated from 2205 samples from 1273 inpatients (ITU: 1338 samples, 784 individuals; Gerontology: 134 samples, 72 individuals; Trauma: 733 samples, 417 individuals) which were spa-typed and analysed here.
Isolation of S. aureusand DNA extraction
Each nasal swab was placed in 5% NaCl enrichment broth (E and O Laboratories) and incubated overnight at 37°C. A loopful of enrichment broth was sub-cultured onto SaSelect chromogenic agar (Bio-Rad) and incubated at 37°C overnight. Pink/orange colonies regarded as S. aureus were positively identified using a Prolex™ Staph Xtra Latex Kit (Pro-Lab Diagnostics) and catalase, DNAse and tube coagulase tests. Methicillin resistance was tested on columbia agar with 5.0% salt (Oxoid) with BBL™ Sensi-Disc™ 1 μg Oxacillin discs (BD).
Mixed glycerol stocks of S. aureus cultures were prepared by suspending several loopfuls of bacteria taken by sweeping across the SaSelect plate in 1.5 ml of saline (E and O Laboratories) with 200 μl of 45% glycerol for storage at −80°C. Taking a sweep across the plate rather than picking a single colony for glycerol stocks allowed us to maintain the genetic diversity of nasal strains in the sample for later analyses.
Crude S. aureus DNA extracts (‘boilates’) used for spa-typing were made from mixed glycerol stocks revived on SaSelect plates. Using a 1 mm loop, a small amount of bacteria was emulsified into 60 μl of Tris-EDTA (TE) buffer (Sigma-Aldrich), then heated in a thermocycler at 99.9°C for 10 minutes and centrifuged at 13,200 × g for 2 minutes. 40 μl of supernatant was removed without disturbing the pellet and stored at −20°C for use as a PCR template.
A staged spa-typing protocol was developed to enable identification of multiple-strain colonization on a large-scale . The polymorphic X region of the protein A gene (spa) was amplified with primers 1095 F: 5′-AGACGATCCTTCGGTGAGC-3′ and 1517R: 5′-GCTTTTGCAATGTCATTTACTG-3′ [28, 29]. PCR reactions consisted of 0.25 mM dNTPs (Qiagen), 0.5 U of GoTaq Flexi DNA Polymerase (Promega), Colorless GoTaq Flexi Buffer, 2.5 mM of magnesium chloride and 0.25 μM of primers in a volume of 10 μl. PCR conditions were 94°C for 2 min; 35 cycles each of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 5 min. PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter).
Samples were sequenced with the same primers as used in PCR. Sequencing reactions used BigDye v3.1 sequencing mix (Applied Biosystems) and were cycled using 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min. Products were purified with Agencourt CleanSEQ beads (Beckman Coulter) and separated on an ABI 3730 DNA Analyzer (Applied Biosystems).
Chromatograms were analyzed using Ridom StaphType v2.0.3 software (Ridom GmbH). The relationships between spa-types were investigated using the BURP clustering algorithm  incorporated into Ridom StaphType.
Identification of rearrangements in spa-gene
To identify rearrangements in the spa-gene which do not affect standard spa-typing, a subset of 32 community samples and 67 bacteraemia samples were sequenced with both spa-3 F/spa-3R and spaT3-F/1517R sets of primers. 67 bacteraemia samples were randomly selected from previously existed collection of hospital invasive isolates.
Sequences were analysed using ProSeq v3.2 (http://dps.plants.ox.ac.uk/sequencing/proseq.htm).
Example sequences for each type of the spa-gene variant have been deposited in the GenBank under the accession numbers JX912490 to JX912498.
Fisher’s exact test, Chi square test and 5x2 exact test were used to compare categorical variables between groups. P values <0.05 were considered statistically significant.
Results and discussion
Identification of rearrangements in the spa-gene
Within two large longitudinal studies of S. aureus carriage in the community (3905 isolates)  and hospital (2205 isolates)  several non-typeable S. aureus strains were identified using standard spa-primers (1095 F/1517R) . Isolates from both studies were spa-typed using a staged protocol, developed to resolve single- and multiple-strain colonization . According to the protocol, spa-sequences were classified as follows: (i) clean sequence traces were interpreted as single strain colonisation, (ii) mixed sequence traces, characterised by distinct double peaks, were interpreted as putative multiple strain colonization, and (iii) unreadable sequence traces represented failed samples, which were retyped. Samples with mixed sequence traces were further resolved by isolating 12 individual colonies; if typing of individual colonies failed, strains were considered non-typeable with standard primers.
Sequence traces of non-typeable samples showed either complete lack of amplification, or mixed sequence traces from both DNA boilates of mixed glycerol stock and of 12 individual colonies. As previously shown , non-typeability of S. aureus strains can be attributed to deletions in the spa-gene, explaining the lack of amplification in some of our samples. However the persistence of mixed sequence traces that could not be resolved by typing individual colonies indicated the presence of other types of spa-gene rearrangements.
With the novel spaT3-F/1517R primer set we were able to type 100% of samples that could not be spa-typed using the standard current set of primers (denoted “formerly non-typeable”).
As expected, non-typeable samples that did not amplify with the standard set of primers had one of two types of deletions affecting the binding site for the original forward primer. Deletion E (174 bp) was previously described by Baum at al. in a clinical S. aureus strain . Deletion G (63 bp) is a novel deletion always paired with insertion B (63 bp) (Figure 3). Non-typeable samples with persistent mixed sequence traces revealed the presence of the insertion C2 (174 bp) (Figure 3). This insertion contains additional binding sites for the spaT3-F and original spa-forward primer, producing two PCR products and distinct double peaks in sequence traces when sequenced with the original spa-forward primer. Sequencing from the reverse primer (1517R) produced clean sequence traces without double peaks.
Surprisingly, in some samples that did not amplify with the standard primer set we found rearrangements represented by deletion A (357 bp) and deletion D/insertion A (174 bp/10 bp) that do not affect the position of the standard forward primer. To investigate the presence of deletions that do not affect spa-typing and therefore can remain unnoticed, we sequenced the whole spa-gene from 32 community carriage and 67 bacteraemia isolates chosen at random from the previously spa-typed collection. We found four novel deletions, deletion D (174 bp) in both bacteraemia and community strains, deletion L (183 bp) only in community strains, deletion H (705 bp) and deletion I/insertion C1 (531 bp/ 174 bp) only in bacteraemia isolates (Figure 3). The largest deletions of three to four IgG-binding domains were found only in S. aureus bacteraemia strains.
Therefore, the presence of different types of deletions and insertions in the spa-gene, identified by spaT3-F/1517R primers, demonstrates that S. aureus colonization/infection is highly complex. People may have a single strain without rearrangements, with deletions that do not affect spa-typing, or with rearrangements that do affect spa-typing. Alternatively, they may carry multiple strains without deletions in any strain, with ‘hidden’ deletions that do not affect spa-typing in one or more strains, or with rearrangements that do affect spa-typing in one or more strains.
Prevalence of spa-gene rearrangements in community and hospital strains
S. aureus isolates with and without different types of rearrangement in the spa -gene in community and inpatient samples: formerly non-typeable isolates
Pure without deletions/insertions or with hidden deletions3
Mixed with or without deletions and/or rearrangements4
Formerly non-typeable: i.e. pure with rearrangements affecting standard spa-typing
1.8% (from total)
1.8% (from total)
0.6% (from total)
0.7% (from total)
27.9% (from 12 picks)
7.4% (from12 picks)
9.3% (from 12 picks)
7.3% (from 12 picks)
S. aureus isolates with and without different types of rearrangement in the spa -gene in community and inpatient samples: isolates with hidden deletions
Total strains without deletions/insertions or with only hidden deletions investigated
Hidden deletions found
Thus up to 13% of S. aureus carriers could, at some point, be colonized with a strain that has deletions/insertions in the IgG-binding region of the spa-gene, 2% carrying completely ‘non-typeable’ strains.
Spa-gene rearrangements lead mixed S. aureuscolonization in humans to be underestimated
The staged spa-typing protocol allowed us to detect the simultaneous presence of two or more strains in 11% of S. aureus carriers. However, the presence of deletions that affect spa-typing in one or more strains within the mixture complicates the typing process and leads to underestimation of the prevalence of multiple colonization and number of strains involved.
Spa -typing of S. aureus strains from a single individual AE with two sets of primers: standard primers 1095 F/1517R and novel primers spaT3-F/1517R
Time points, months
DNA prep (mixed boilate)
12 single colony picks2
The limitations of the conventional spa-typing protocol make it impossible to identify and type S. aureus strains with rearrangements in the spa-gene in individuals with multiple strain colonization. The staged spa-typing protocol allows us to resolve cases of mixed strain colonization with deletions in one or more strains. Even 12 single colony picks could not always identify the presence of low-frequency strains with deletions, illustrating the even greater challenge of estimating the proportion of non-typeable strains within mixed colonization. Thus diversity in colonizing and infecting strains is inevitably underestimated.
Inpatients’ strains can acquire deletions in the spa-gene
We also found that S. aureus strains can acquire deletions in the spa-gene during inpatients’ hospital admission. Such acquisition of deletions was never observed for longitudinal carriage strains from individuals in the community, with those deletions observed being present from the first time the strain carrying the deletion was identified (Additional file 1: Table S1).
Individuals who acquired a deletion in the S. aureus spa -gene during their hospital admission
Date swab taken
The repetitive nature of the spa-gene makes it unstable and highly prone to internal rearrangements, which in bacteria occur via either RecA-dependent or RecA-independent recombination [31–33]. These rearrangements might have positive or negative effects as protein A is an important virulence factor that plays a central role in S. aureus defence against the host immune response. There is new evidence that the antibiotic ciprofloxacin increases the intrachromosomal DNA recombination rate in Escherichia coli. Other antibiotics might potentially have similar effects, yet undiscovered. Taking into account that the three inpatients who acquired deletions during their stay at the hospital had been taking specific antibiotics for a long time or a wide range of antibiotics for a short period, including ciprofloxacin, it is possible that antibiotic pressure might be one factor that drives genetic rearrangements in the S. aureus protein A gene. However, we also cannot exclude the possibility that these deletions may have been present already at low frequency, and undetected, before increasing to become the majority variant (rather than being acquired de novo). Nevertheless this scenario also would support antibiotics playing a role in emergence of deletions to detectable levels.
In the community, most individuals colonized by S. aureus strains carry them without displaying any symptoms. However, when some of them became invasive, the change of habitat, for example on a background of antibiotic pressure, might promote acquisition of rearrangements in the spa-gene that might be advantageous in new environment even if they lead to loss or change of protein function. As shown previously, progress of infectious disease is typically associated with acquisition of mutations or rearrangements in the pathogen, many of which lead to loss of function in a number of genes .
Deletions appear to be over-represented in clonal lineages related to livestock
Spa -types and groups in which deletions/insertions in the spa -gene were observed
Individuals with deletions, no. (column %)
Hidden deletions not affecting spa-typing (no.)
Deletions/insertions affecting spa-typing (no.)
Individuals with deletions affecting spa-typing/total individuals with this spa-type
delE (1); insC2 (1)
delD (1); delA (1)
Any clusters of related spa-types with a higher prevalence of rearrangements affecting spa-typing (delE, delG-insB or insC2) would be likely to be underrepresented, or even missing, in the majority of studies based on routine spa-typing protocols. To test this hypothesis, we compared the proportion of individuals with and without rearrangements affecting spa-typing in the four groups of spa-types and the singleton that we found had one or more rearrangements affecting spa-typing (5 × 2 exact test, Table 5). In group 1 35% of strains were affected by these rearrangements, a significantly higher proportion compared with the 1-4% in other groups or the singleton t530 (p < 0.0001). Therefore, spa-type t571 and its closely related variants such as t3085 may well be underrepresented in most S. aureus studies based on spa-typing, as they could not be typed with the standard set of primers when common deletions are present.
Interestingly, spa-type t571 belongs to clonal lineage ST398 that contains MRSA and MSSA strains common among livestock. Spa-type t571 is closely related to type t011 and t034, all most commonly associated with pigs [36–40]. These spa-types have been found less commonly in dogs, cats and horses, and occasionally in cattle and poultry [41, 42]. Large-scale screening of pigs  showed that 60% of them carried t034, 14% t1255 and 1.5% t571. Although ST398-associated spa-types have been rarely found among the general human population, they have been found more commonly in farmers working with pigs [36, 37]. Veterinary personnel and pet owners are also more likely to carry these animal-related types . Recent studies have also reported the emergence of livestock-associated MRSA clones of S. aureus ST398 causing bacteraemias in humans, supporting animal-independent transmission of such strains between humans [44, 45].
It is unclear why ST398 S. aureus strains commonly found in livestock frequently develop deletions in the IgG-binding part of protein A gene after transmission to humans. One possible explanation is that this might be a part of S. aureus strain adaptation to a different immune background where protein A plays a major role [7, 8, 12]. Another explanation might be that the livestock associated strains have more rearrangements in the spa-gene prior the transmission to humans due to high level of antibiotic exposure in food-animal production [46–48]. Nevertheless, our findings highlight the potential for these strains to have been substantially under-represented in epidemiological studies to date, and for strains formerly not-typeable using standard methods to be a source of bias.
Spa-typing is a common technique used to classify S. aureus strains in clinical practice (eg outbreak management) and research. Rearrangements in the IgG-binding region of the spa-gene make strains “non-typeable” with commonly used primers. Using a novel primer, we typed 100% of samples and identified eight novel spa-gene variants, plus one previously described; three of these rearrangements cause strains to be designated as “non-typeable” using current spa-typing methods.
Spa-typing of 6110 S. aureus isolates showed that 1.8% of samples from 1.8% community carriers and 0.6% of samples from 0.7% inpatients were formerly non-typeable. We also found evidence of mixed colonization with strains with and without gene rearrangements, and estimated that up to 13% of carriers are colonized with “hidden” S. aureus with deletions/insertions in the IgG-binding region at some point. Using standard primers therefore underestimates spa-type diversity. We also found evidence of inpatients acquiring spa-gene deletions de novo during a hospital admission, suggesting that antibiotic pressure might be one factor driving genetic rearrangements in the S. aureus protein A gene. Finally, we found that deletions formerly causing strains to be designated as “non-typeable” were over-represented in clonal lineages related to livestock, indicating that these may well be have been underrepresented in most S. aureus studies. This new improved spa-typing protocol therefore enables previously overlooked S. aureus strains to be typed and therefore contribute to our understanding of diversity, carriage and transmission of S. aureus strains in community and hospitals.
The authors wish to thank Dr. Teresa Street for discussion of the laboratory results, Dr. Kate Dingle for the comments on the manuscript, Ms. Alison Vaughan and Mr. David Griffiths for their assistance in the laboratory.
This study was supported by the Oxford NIHR Biomedical Research Centre and the UKCRC Modernising Medical Microbiology Consortium, with the latter funded under the UKCRC Translational Infection Research Initiative supported by Medical Research Council, Biotechnology and Biological Sciences Research Council and the National Institute for Health Research on behalf of the Department of Health (Grant G0800778) and The Wellcome Trust (Grant 087646/Z/08/Z).
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