Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids-who's mimicking whom?

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.

Corrigendum: Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

[This corrects the article DOI: 10.3389/fmicb.2018.02664.].

Fitness costs associated with carriage of a large staphylococcal plasmid are reduced by subinhibitory concentrations of antiseptics.

Staphylococcus aureus carries a collection of mobile genetic elements that often harbor virulence and antimicrobial resistance genes. Since the introduction of antibiotics, plasmids have become a major genetic element responsible for the distribution of antimicrobial resistance. Under antimicrobial selection, resistance plasmids are maintained within bacterial populations as a means to ensure survival. However, in the absence of selection, large plasmids can be lost due to the fitness costs associated with harboring these genetic elements. pC02 is a previously identified multidrug resistance, conjugative plasmid that is found in S. aureus. In addition to antibiotic resistance, pC02 also carries genes known to be associated with antiseptic resistance. Among these, we previously characterized the contribution of qacA to pC02 mediated reduced chlorhexidine susceptibility. Herein, we demonstrate that pC02 also mediates triclosan resistance, likely due to the presence of fabI, a known triclosan resistance gene. Moreover, we demonstrate that conjugative transfer of pC02 increases triclosan resistance in recipient cells. Competition assays demonstrated a fitness cost associated with carriage of the large pC02 plasmid. However, subinhibitory concentrations of either chlorhexidine or triclosan abrogated this fitness cost. Given the widespread use of these antiseptics, both of which accumulate in wastewater and other environmental reservoirs, indiscriminate use of antiseptics likely imposes a constant selective pressure that promotes maintenance of antimicrobial resistance factors within S. aureus.

Systematic Analysis of Efflux Pump-Mediated Antiseptic Resistance in Staphylococcus aureus Suggests a Need for Greater Antiseptic Stewardship.

Staphylococcus aureus-associated infections can be difficult to treat due to multidrug resistance. Thus, infection prevention is critical. Cationic antiseptics, such as chlorhexidine (CHX) and benzalkonium chloride (BKC), are liberally used in health care and community settings to prevent infection. However, increased administration of antiseptics has selected for S. aureus strains that show reduced susceptibilities to cationic antiseptics. This increased resistance has been associated with carriage of specific efflux pumps (QacA, QacC, and NorA). Since prior published studies focused on different strains and on strains carrying only a single efflux gene, the relative importance of these various systems to antiseptic resistance is difficult to ascertain. To overcome this, we engineered a collection of isogenic S. aureus strains that harbored norA, qacA, and qacC, individually or in combination. MIC assays showed that qacA was associated with increased resistance to CHX, cetrimide (CT), and BKC, qacC was associated with resistance to CT and BKC, and norA was necessary for basal-level resistance to the majority of tested antiseptics. When all three pumps were present in a single strain, an additive effect was observed in the MIC for CT. Transcriptional analysis revealed that expression of qacA and norA was significantly induced following exposure to BKC. Alarmingly, in a strain carrying qacA and norA, preexposure to BKC increased CHX tolerance. Overall, our results reveal increased antiseptic resistance in strains carrying multiple efflux pumps and indicate that preexposure to BKC, which is found in numerous daily-use products, can increase CHX tolerance.IMPORTANCES. aureus remains a significant cause of disease within hospitals and communities. To reduce the burden of S. aureus infections, antiseptics are ubiquitously used in our daily lives. Furthermore, many antiseptic compounds are dual purpose and are found in household products. The increased abundance of antiseptic compounds has selected for S. aureus strains that carry efflux pumps that increase resistance to antiseptic compounds; however, the effect of carrying multiple pumps within S. aureus is unclear. We demonstrated that an isogenic strain carrying multiple efflux pumps had an additive resistance phenotype to cetrimide. Moreover, in a strain carrying qacA and norA, increased chlorhexidine tolerance was observed after the strain was preexposed to subinhibitory concentrations of a different common-use antiseptic. Taken together, our findings demonstrate cooperation between antiseptic resistance efflux pumps and suggest that their protective phenotype may be exacerbated by priming with subinhibitory concentrations of household antiseptics.

Conjugative and replicative biology of the Staphylococcus aureus antimicrobial resistance plasmid, pC02.

Genetic transfer among bacteria propels rapid resistance to antibiotics and decreased susceptibility to antiseptics. Staphylococcus aureus is a common culprit of hospital and community acquired infections, and S. aureus plasmids have been shown to carry a multitude of antimicrobial resistance genes. We previously identified a novel conjugative, multidrug resistance plasmid, pC02, from the clinical S. aureus isolate C02. This plasmid contained the chlorhexidine resistance gene qacA, and we were able to demonstrate that conjugative transfer of pC02 imparted decreased chlorhexidine susceptibility to recipient strains. In silico sequence analysis of pC02 suggested that the plasmid is part of the pWBG749-family of conjugative plasmids and that it contains three predicted origins of transfer (oriT), two of which we showed were functional and could mediate plasmid transfer. Furthermore, depending on which oriT was utilized, partial transfer of pC02 was consistently observed. To define the ability of the pC02 plasmid to utilize different oriT sequences, we examined the mobilization ability of nonconjugative plasmid variants that were engineered to contain a variety of oriT family inserts. The oriT-OTUNa family was transferred at the highest frequency; additional oriT families were also transferred but at lower frequencies. Plasmid stability was examined, and the copy number of pC02 was defined using droplet digital PCR (ddPCR). pC02 was stably maintained at approximately 4 copies per cell. Given the conjugative plasticity of pC02, we speculate that this plasmid could contribute to the spread of antimicrobial resistance across Staphylococcal strains and species.

Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

Bacterial Etiology and Risk Factors Associated with Cellulitis and Purulent Skin Abscesses in Military Trainees.

Military trainees are at high risk for skin and soft-tissue infections (SSTIs). Although Staphylococcus aureus is associated with purulent SSTI, it is unclear to what degree this pathogen causes nonpurulent cellulitis. To inform effective prevention strategies and to provide novel insights into SSTI pathogenesis, we aimed to determine the etiology of SSTI in this population. We conducted a prospective observational study in US Army Infantry trainees with SSTI (cutaneous abscesses and cellulitis) from July 2012 through December 2014. We used standard microbiology, serology, and high-throughput sequencing to determine the etiology of SSTI. Furthermore, we compared purported risk factors as well as anatomic site colonization for S. aureus. Among 201 SSTI cases evaluated for SSTI risk factors, cellulitis was associated with lower extremity blisters (P = 0.01) and abscess was associated with methicillin-resistant S. aureus (MRSA) colonization (P<0.001). Among the 22 tested cellulitis cases that were part of the microbiome analysis, only 1 leading edge aspirate was culturable (Coagulase-negative Staphylococcus). Microbiome evaluation of aspirate specimens demonstrated that Rhodanobacter terrae was the most abundant species (66.8% average abundance), while abscesses were dominated by S. aureus (92.9% average abundance). Although abscesses and cellulitis share the spectrum of clinical SSTI, the bacterial etiologies as determined by current technology appear distinct. Furthermore, the presence of atypical bacteria within cellulitis aspirates may indicate novel mechanisms of cellulitis pathogenesis. CLINICAL TRIALS REGISTRATION: NCT01105767.

Stx-Producing Shigella Species From Patients in Haiti: An Emerging Pathogen With the Potential for Global Spread.

Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.