Davida S. Smyth

Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections

ABSTRACT Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolates genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Polyphyletic Emergence of Linezolid-Resistant Staphylococci in the United States

ABSTRACT Since the year 2000, linezolid has been used in the United States to treat infections caused by antimicrobial-resistant Gram-positive cocci. At present, linezolid-resistant (Linr) Staphylococcus aureus and Staphylococcus epidermidis strains are rare and the diversity of their genetic backgrounds is unknown. We performed sequence-based strain typing and resistance gene characterization of 46 Linr isolates that were collected from local and national sources between the years 2004 and 2007. Resistance was found to occur in at least three clonal complexes (CCs; lineages) of S. aureus and in at least four subclusters of a predominant, phylogenetically unstable CC of S. epidermidis. New candidate resistance mutations in 23S rRNA and the L4 riboprotein were identified among the S. epidermidis isolates. These findings suggest that linezolid resistance has emerged independently in multiple clones of S. aureus and with a variety of ribosomal mutations in multiple clones of S. epidermidis.

Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.

Cross-species spread of SCCmec IV subtypes in staphylococci.

Staphylococcal chromosomal cassette mec (SCCmec) is a mobile genetic element that carries resistance genes for beta-lactam antibiotics. Coagulase-negative staphylococci, such as S. epidermidis, are thought to be a reservoir of diverse SCCmec elements that can spread to the most virulent staphylococcal species, S. aureus, but very little is known about the extent of cross-species spread of these elements in natural populations or their dynamics in different species. We addressed these questions using a sample of 86 S. aureus and S. epidermidis isolates with SCCmec type IV that were collected from a single hospital over a period of 6 months. To subtype SCCmec IV, we used multiplex PCR to detect structural variations and we used sequences from a fragment of the ccrB gene and from the dru repeats to detect additional variations. Multiplex PCR had significantly lower typeability than ccrB:dru sequencing, due to more nontypeable isolates among S. epidermidis. No statistically significant differences in diversity were detected by subtyping method or species. Interestingly, while only 4 of 24 subtypes (17%) were shared between species, these so-called shared subtypes represented 58 of 86 isolates (67%). The shared subtypes differed significantly between species in their frequencies. The shared subtypes were also significantly more concordant with genetic backgrounds in S. aureus than in S. epidermidis. Moreover, the shared subtypes had significantly higher minimum inhibitory concentrations to oxacillin in S. aureus than in S. epidermidis. This study has identified particular SCCmec IV subtypes with an important role in spreading beta-lactam resistance between species, and has further revealed some species differences in their abundance, linkage to genetic background, and antibiotic resistance level.

Identification of a novel transposon (Tn6072) and a truncated staphylococcal cassette chromosome mec element in methicillin-resistant Staphylococcus aureus ST239.

ABSTRACT A novel composite transposon (Tn6072) resembling staphylococcal cassette chromosome mercury (SCCHg) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn6072 is homologous to the 5′ region of SCCHg found in staphylococcal cassette chromosome mec (SCCmec) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCCHg has previously been reported to integrate downstream of orfX, at the same chromosomal location as SCCmec. Tn6072, by contrast, is demarcated by two IS431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn6072, demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCCmec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCCmec background. BK16704 possesses an SCCmec 3A.1.4 structure, with an IS256 insertion downstream of the right chromosomal junction. In contrast, the SCCmec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS256 element. This structure, tentatively named “ψSCCmec16691,” confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group.

Microbiological and Genotypic Analysis of Methicillin-Resistant Staphylococcus aureus Bacteremia

ABSTRACT In a recent landmark trial of bacteremia caused by methicillin-resistant Staphylococcus aureus (MRSA) isolates, vancomycin MICs were ≥1 μg/ml for only 16% of the isolates, and accessory gene regulator (agr) function as measured by delta-hemolysin activity was absent or reduced in only 28.1% of the isolates. This clinical study did not capture a population of MRSA isolates predictive of vancomycin treatment failure.

Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature.

A previous study carried out by the National Food Centre in Dublin on bacterial contamination of Irish domestic refrigeration systems revealed that 41% were contaminated with Staphylococcus aureus. One hundred fifty-seven S. aureus isolates were screened by multiplex PCR analysis for the presence of 15 staphylococcal enterotoxin and enterotoxin-like genes (sea-see, seg-sei, selj-selo, and selq) and the toxic shock toxin superantigen tst gene. Of the refrigerator isolates, 64.3% possessed more than one staphylococcal enterotoxin or staphylococcal enterotoxin-like gene. All bar one of the 101 staphylococcal enterotoxin or staphylococcal enterotoxin-like gene-positive strains possessed the egc locus bearing the seg, sei, selm, seln, and selo genes. Twelve random amplified polymorphic DNA (RAPD) types accounted for 119 (75.8%) of the strains, two of these types accounting for 25 (RAPD type 1, 15.9%) and 52 (RAPD type 5, 33.1%), respectively. All of the RAPD type 5 isolates possessed the egc gene cluster only. The RAPD type 5 amplicon profile was identical to that of S. aureus isolates associated with osteomyelitis in broiler chickens in Northern Ireland that also possessed the egc locus only. However, the RAPD type 5 domestic refrigerator and chicken isolates differed in penicillin G sensitivity, production of Protein A and staphylokinase, and crystal violet agar growth type. These findings highlight that the average Irish household refrigerator harbors potential enterotoxin-producing S. aureus that may or may not be of animal origin and, accordingly, is a potential reservoir for staphylococcal food poisoning.

Phage-inducible islands in the Gram-positive cocci

The SaPIs are a cohesive subfamily of extremely common phage-inducible chromosomal islands (PICIs) that reside quiescently at specific att sites in the staphylococcal chromosome and are induced by helper phages to excise and replicate. They are usually packaged in small capsids composed of phage virion proteins, giving rise to very high transfer frequencies, which they enhance by interfering with helper phage reproduction. As the SaPIs represent a highly successful biological strategy, with many natural Staphylococcus aureus strains containing two or more, we assumed that similar elements would be widespread in the Gram-positive cocci. On the basis of resemblance to the paradigmatic SaPI genome, we have readily identified large cohesive families of similar elements in the lactococci and pneumococci/streptococci plus a few such elements in Enterococcus faecalis. Based on extensive ortholog analyses, we found that the PICI elements in the four different genera all represent distinct but parallel lineages, suggesting that they represent convergent evolution towards a highly successful lifestyle. We have characterized in depth the enterococcal element, EfCIV583, and have shown that it very closely resembles the SaPIs in functionality as well as in genome organization, setting the stage for expansion of the study of elements of this type. In summary, our findings greatly broaden the PICI family to include elements from at least three genera of cocci.