Dynamics of the Oral Microbiome During Initial Military Training at Fort Benning, Georgia.

INTRODUCTION: Military trainees are at increased risk for infectious disease outbreaks because of the unique circumstances of the training environment (e.g., close proximity areas and physiologic/psychologic stress). Standard medical countermeasures in military training settings include routine immunization (e.g., influenza and adenovirus) as well as chemoprophylaxis [e.g., benzathine penicillin G (Bicillin) for the prevention of group A streptococcal disease] for pathogens associated with outbreaks in these settings. In a population of U.S. Army Infantry trainees, we evaluated changes in the oral microbiome during a 14-week military training cycle. MATERIALS AND METHODS: Trainees were enrolled in an observational cohort study in 2015-2016. In 2015, Bicillin was administered to trainees to ameliorate the risk of group A Streptococcus outbreaks, whereas in 2016, trainees did not receive a Bicillin inoculation. Oropharyngeal swabs were collected from participants at days 0, 7, 14, 28, 56, and 90 of training. Swabs were collected, flash frozen, and stored. DNA was extracted from swabs, and amplicon sequencing of the 16s rRNA gene was performed. Microbiome dynamics were evaluated using the QIIME 2 workflow along with DADA2, SINA with SILVA, and an additional processing in R. RESULTS: We observed that microbiome samples from the baseline (day 0) visit were distinct from one another, whereas samples collected on day 14 exhibited significant microbiome convergence. Day 14 convergence was coincident with an increase in DNA sequences associated with Streptococcus, though there was not a significant difference between Streptococcus abundance over time between 2015 and 2016 (P = .07), suggesting that Bicillin prophylaxis did not significantly impact overall Streptococcus abundance. CONCLUSIONS: The temporary convergence of microbiomes is coincident with a rise in communicable infections in this population. The dynamic response of microbiomes during initial military training supports similar observations in the literature of transient convergence of the human microbiome under cohabitation in the time frame including in this experiment. This population and the associated longitudinal studies allow for controlled studies of human microbiome under diverse conditions.

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease.

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

Nasal microbiota evolution within the congregate setting imposed by military training.

The human microbiome is comprised of a complex and diverse community of organisms that is subject to dynamic changes over time. As such, cross-sectional studies of the microbiome provide a multitude of information for a specific body site at a particular time, but they fail to account for temporal changes in microbial constituents resulting from various factors. To address this shortcoming, longitudinal research studies of the human microbiome investigate the influence of various factors on the microbiome of individuals within a group or community setting. These studies are vital to address the effects of host and/or environmental factors on microbiome composition as well as the potential contribution of microbiome members during the course of an infection. The relationship between microbial constituents and disease development has been previously explored for skin and soft tissue infections (SSTIs) within congregate military trainees. Accordingly, approximately 25% of the population carries Staphylococcus aureus within their nasal cavity, and these colonized individuals are known to be at increased risk for SSTIs. To examine the evolution of the nasal microbiota of U.S. Army Infantry trainees, individuals were sampled longitudinally from their arrival at Fort Benning, Georgia, until completion of their training 90 days later. These samples were then processed to determine S. aureus colonization status and to profile the nasal microbiota using 16S rRNA gene-based methods. Microbiota stability differed dramatically among the individual trainees; some subjects exhibited great stability, some subjects showed gradual temporal changes and some subjects displayed a dramatic shift in nasal microbiota composition. Further analysis utilizing the available trainee metadata suggests that the major drivers of nasal microbiota stability may be S. aureus colonization status and geographic origin of the trainees. Nasal microbiota evolution within the congregate setting imposed by military training is a complex process that appears to be affected by numerous factors. This finding may indicate that future campaigns to prevent S. aureus colonization and future SSTIs among high-risk military trainees may require a 'personalized' approach.

The Antimicrobial Potential of the Neem Tree Azadirachta indica.

Azadirachta indica (A. Juss), also known as the neem tree, has been used for millennia as a traditional remedy for a multitude of human ailments. Also recognized around the world as a broad-spectrum pesticide and fertilizer, neem has applications in agriculture and beyond. Currently, the extensive antimicrobial activities of A. indica are being explored through research in the fields of dentistry, food safety, bacteriology, mycology, virology, and parasitology. Herein, some of the most recent studies that demonstrate the potential of neem as a previously untapped source of novel therapeutics are summarized as they relate to the aforementioned research topics. Additionally, the capacity of neem extracts and compounds to act against drug-resistant and biofilm-forming organisms, both of which represent large groups of pathogens for which there are limited treatment options, are highlighted. Updated information on the phytochemistry and safety of neem-derived products are discussed as well. Although there is a growing body of exciting evidence that supports the use of A. indica as an antimicrobial, additional studies are clearly needed to determine the specific mechanisms of action, clinical efficacy, and in vivo safety of neem as a treatment for human pathogens of interest. Moreover, the various ongoing studies and the diverse properties of neem discussed herein may serve as a guide for the discovery of new antimicrobials that may exist in other herbal panaceas across the globe.

The Role of Microorganisms in Appendiceal Pseudomyxoma Peritonei: A Review.

Pseudomyxoma peritonei (PMP) is a rare clinical syndrome. It originates from neoplasms of the appendix and leads to the formation of peritoneal implants and the accumulation of mucinous ascites. PMP represents a spectrum of low to high-grade disease. Despite aggressive management, many PMP patients recur, leading to debilitating symptoms and few treatment options. Therefore, scientists have continued to look for ways to improve treatment and further understand disease pathogenesis. Microorganisms were previously hypothesized to play a role in PMP progression and development. Hence, antibacterial treatment was suggested by some authors, but the data were limited. In this paper, we review the current data on the role of bacteria in PMP, discuss the significance, and suggest possible solutions to the inherent challenges in these studies. Given the limitations of the discussed studies, we remain skeptical about introducing novel antibacterial treatment into clinical practice at this time; however, the available data are valuable and indicate that more research into the molecular mechanisms of PMP is needed.

Interplay between Amoxicillin Resistance and Osmotic Stress in Helicobacter pylori.

Rising antibiotic resistance rates are a growing concern for all pathogens, including Helicobacter pylori. We previously examined the association of specific mutations in PBP1 with amoxicillin resistance and fitness in H. pylori and found that V374L and N562Y mutations were associated with resistance, but also resulted in fitness defects. Furthermore, we found that hyperosmotic stress differentially altered the fitness of strains bearing these mutations; survival of the V374L strain was decreased by hyperosmotic stress, but the N562Y strain showed increased cell survival relative to that of wild-type G27. The finding that amoxicillin-resistant strains show environmentally dictated changes in fitness suggests a previously unexplored interaction between amoxicillin resistance and osmotic stress in H. pylori. Here, we further characterized the interaction between osmotic stress and amoxicillin resistance. Wild-type and isogenic PBP1 mutant strains were exposed to amoxicillin, various osmotic stressors, or combined antibiotic and osmotic stress, and viability was monitored. While subinhibitory concentrations of NaCl did not affect H. pylori viability, the combination of NaCl and amoxicillin resulted in synergistic killing; this was true even for the antibiotic-resistant strains. Moreover, similar synergy was found with other beta-lactams, but not with antibiotics that did not target the cell wall. Similar synergistic killing was also demonstrated when KCl was utilized as the osmotic stressor. Conversely, osmolar equivalent concentrations of sucrose antagonized amoxicillin-mediated killing. Taken together, our results support a previously unrecognized interaction between amoxicillin resistance and osmotic stress in H. pylori. These findings have interesting implications for the effectiveness of antibiotic therapy for this pathogen. IMPORTANCE Rising antibiotic resistance rates in H. pylori are associated with increased rates of treatment failure. Understanding how stressors impact antibiotic resistance may shed light on the development of future treatment strategies. Previous studies found that mutations in PBP1 that conferred resistance to amoxicillin were also associated with a decrease in bacterial fitness. The current study demonstrated that osmotic stress can enhance beta lactam-mediated killing of H. pylori. The source of osmotic stress was found to be important for these interactions. Given that relatively little is known about how H. pylori responds to osmotic stress, these findings fill important knowledge gaps on this topic and provide interesting implications for the effectiveness of antibiotic therapy for this pathogen.

In vitro antibacterial activity of nimbolide against Helicobacter pylori.

ETHNOPHARMACOLOGICAL RELEVANCE: Nimbolide is one of hundreds of phytochemicals that have been identified within the neem tree (Azadirachta indica A. Juss). As an evergreen tree native to the Indian subcontinent, components of the neem tree have been used for millennia in traditional medicine to treat dental, gastrointestinal, urinary tract, and blood-related ailments, ulcers, headaches, heartburn, and diabetes. In modern times, natural oils and extracts from the neem tree have been found to have activities against a variety of microorganisms, including human pathogens. AIM OF THE STUDY: Helicobacter pylori, a prevalent gastric pathogen, shows increasing levels of antibiotic resistance. Thus, there is an increasing demand for novel therapeutics to treat chronic infections. The in vitro activity of neem oil extract against H. pylori was previously characterized and found to be bactericidal. Given the numerous phytochemicals found in neem oil extract, the present study was designed to define and characterize specific compounds showing bactericidal activity against H. pylori. MATERIALS AND METHODS: Azadirachtin, gedunin, and nimbolide, which are all common in neem extracts, were tested for antimicrobial activity; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for nine strains of H. pylori. The specific properties of nimbolide were further characterized against H. pylori strain G27. Bactericidal kinetics, reversibility, effectiveness at low pH, and activity under bacteriostatic conditions were examined. The hemolytic activity of nimbolide was also measured. Finally, neem oil extract and nimbolide effectiveness against H. pylori biofilms were examined in comparison to common antibiotics used to treat H. pylori infection. RESULTS: Nimbolide, but not azadirachtin or gedunin, was effective against H. pylori; MICs and MBCs against the nine tested strains ranged between 1.25-5 µg/mL and 2.5-10 µg/mL, respectively. Additionally, neem oil extract and nimbolide were both effective against H. pylori biofilms. Nimbolide exhibited no significant hemolytic activity at biologically relevant concentrations. The bactericidal activity of nimbolide was time- and dose-dependent, independent of active H. pylori growth, and synergistic with low pH. Furthermore, nimbolide-mediated H. pylori cell death was irreversible after exposure to high nimbolide concentrations (80 µg/mL, after 2 h of exposure time and 40 µg/mL after 8 h of exposure). CONCLUSIONS: Nimbolide has significant bactericidal activity against H. pylori, killing both free living bacterial cells as well as cells within a biofilm. Furthermore, the lack of hemolytic activity, synergistic activity at low pH and bactericidal properties even against bacteria in a state of growth arrest are all ideal pharmacological and biologically relevant properties for a potential new agent. This study underscores the potential of neem oil extract or nimbolide to be used as a future treatment for H. pylori infection.

Characterization of East-Asian Helicobacter pylori encoding Western EPIYA-ABC CagA.

The polymorphic bacterial oncoprotein, CagA shows geography-dependent variation in the C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs; East-Asian H. pylori isolates carry the ABD type while Western isolates carry the ABC type. In Western isolates, the EPIYA-C motif is sometimes found in multi-copy and this genotype is associated with disease severity. Interestingly, a small number of East-Asian H. pylori isolates have been found to carry Western ABC-type CagA. To gain a better understanding of these unusual isolates, the genomes of four Korean H. pylori clinical isolates carrying ABC-type CagA were sequenced via third generation (Pac-Bio SMRT) sequencing technology. The obtained data were utilized for phylogenetic analysis as well as comparison of additional virulence factors that are known to show geographic-dependent polymorphisms. Three of four isolates indeed belonged to the hpEastAsia group and showed typical East-Asian polymorphism in virulence factors such as homA/B/C, babA/B/C, and oipA. One isolate grouped to HpAfrica and showed typical Western polymorphism of virulence factors such as cagA, homA/B/C, and oipA. To understand the occurrence of the multi-copy EPIYA-C motif genotype in an East-Asian H. pylori background, the Korean clinical isolate, K154 was analyzed; this strain belonged to hpEastAsia but encoded CagA EPIYA-ABCCCC. Based on DNA sequence homology within the CagA multimerization (CM) sequence that flanked the EPIYA-C motifs, we predicted that the number of C motifs might change via homologous recombination. To test this hypothesis, K154 was cultured for one generation and 287 single colonies were analyzed for the number of EPIYA-C motifs using PCR-based screening and DNA sequencing verification. Three out of 284 (1%) single colony isolates showed changes in the number of EPIYA-C motifs in vitro; one isolate increased to five EPIYA-C motifs, one decreased to three EPIYA-C motifs, and one completely deleted the EPIYA-C motifs. The capacity for dynamic changes in the number of EPIYA-C repeats of CagA may play a role in generating important intraspecies diversity in East-Asian H. pylori.

Geographic diversity in Helicobacter pylori oipA genotype between Korean and United States isolates.

Helicobacter pylori outer membrane inflammatory protein A (OipA) was originally named for its role in inducing inflammation in the host, as evidenced by high mucosal IL-8 levels. Expression of OipA is regulated by phase variation of a CT dinucleotide-repeat located in the 5' region of the gene. However, little is known about OipA geographic diversity across isolates. To address this gap, we conducted a large-scale molecular epidemiologic analysis using H. pylori clinical isolates obtained from two geographically distinct populations: Korea and the United States (US). Most Korean isolates (98.7%) possessed two copies of oipA located at two specific loci (A and B) while all US isolates contained only one copy of oipA at locus A. Furthermore, most Korean oipA (94.8%) possessed three or less CT repeats while most US oipA (96.6%) contained five or more CT repeats. Among the two copies, all Korean H. pylori possessed at least one oipA 'on' phase variant while the single copy of oipA in US isolates showed 56.2% 'on' and 43.8% 'off.' Thus, host differences seem to have driven geographic diversification of H. pylori across these populations such that OipA expression in US isolates is still regulated by phase variation with 5 or more CT repeats, while Korean isolates always express OipA; duplication of the oipA combined with a reduction of CT repeats to three or less ensures continued expression. En masse, these findings suggest that diversity in the oipA gene copy number, CT repeats, and phase variation among H. pylori from different populations may confer a benefit in adaptation to particular host populations.

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.

Generation of Isogenic Mutant Strains of Helicobacter pylori.

One important tool in the study of gene function is the construction of mutant strains. Specifically, the construction of isogenic mutant strains imparts researchers with the ability to compare a wild-type strain to a strain that is genetically identical with the exception of the gene of interest. For a bacterial species such as Helicobacter pylori , which is notorious for the genetic heterogeneity seen across isolates, comparisons between isogenic and parental strains control for the genetic variation seen between distinct isolates. This chapter details the construction of a clean gene deletion in which the entire coding region is replaced with a selectable marker. The approach detailed herein allows for the thorough investigation of gene function in the absence of confounding genetic variability.

Gut microbiota and metabolic marker alteration following dietary isoflavone-photoperiod interaction.

Introduction: The interaction between isoflavones and the gut microbiota has been highlighted as a potential regulator of obesity and diabetes. In this study, we examined the interaction between isoflavones and a shortened activity photoperiod on the gut microbiome. Methods: Male mice were exposed to a diet containing no isoflavones (NIF) or a regular diet (RD) containing the usual isoflavones level found in a standard vivarium chow. These groups were further divided into regular (12L:12D) or short active (16L:8D) photoperiod, which mimics seasonal changes observed at high latitudes. White adipose tissue and genes involved in lipid metabolism and adipogenesis processes were analysed. Bacterial genomic DNA was isolated from fecal boli, and 16S ribosomal RNA sequencing was performed. Results: NIF diet increased body weight and adipocyte size when compared to mice on RD. The lack of isoflavones and photoperiod alteration also caused dysregulation of lipoprotein lipase (Lpl), glucose transporter type 4 (Glut-4) and peroxisome proliferator-activated receptor gamma (Pparg) genes. Using 16S ribosomal RNA sequencing, we found that mice fed the NIF diet had a greater proportion of Firmicutes than Bacteroidetes when compared to animals on the RD. These alterations were accompanied by changes in the endocrine profile, with lower thyroid-stimulating hormone levels in the NIF group compared to the RD. Interestingly, the NIF group displayed increased locomotion as compared to the RD group. Conclusion: Together, these data show an interaction between the gut bacterial communities, photoperiod length and isoflavone compounds, which may be essential for understanding and improving metabolic health.

Friend or Foe: Interbacterial Competition in the Nasal Cavity.

Like other microbes that live on or in the human body, the bacteria that inhabit the upper respiratory tract, in particular the nasal cavity, have evolved to survive in an environment that presents a number of physical and chemical challenges; these microbes are constantly bombarded with nutritional fluctuations, changes in humidity, the presence of inhaled particulate matter (odorants and allergens), and competition with other microbes. Indeed, only a specialized set of species is able to colonize this niche and successfully contend with the host's immune system and the constant threat from competitors. To this end, bacteria that live in the nasal cavity have evolved a variety of approaches to outcompete contenders for the limited nutrients and space; broadly speaking, these strategies may be considered a type of "bacterial warfare." A greater molecular understanding of bacterial warfare has the potential to reveal new approaches or molecules that can be developed as novel therapeutics. As such, there are many studies within the last decade that have sought to understand the complex polymicrobial interactions that occur in various environments. Here, we review what is currently known about the age-dependent structure and interbacterial relationships within the nasal microbiota and summarize the molecular mechanisms that are predicted to dictate bacterial warfare in this niche. Although the currently described interactions are complex, in reality, we have likely only scratched the surface in terms of a true understanding of the types of interbacterial competition and cooperation that are thought to take place in and on the human body.

Analysis of fitness costs associated with metronidazole and amoxicillin resistance in Helicobacter pylori.

BACKGROUND: Increasing rates of antibiotic resistance are a major concern for all pathogens, including H. pylori. However, increased resistance often coincides with a decrease in relative fitness of the pathogen in the absence of the antibiotic, raising the possibility that increased resistance can be mitigated for some antibiotics by improved antibiotic husbandry. Therefore, a greater understanding of which types of antibiotic resistance create fitness defects in H. pylori may aid rational treatment strategies. MATERIALS AND METHODS: While a wealth of H. pylori literature reports mutations that correlate with increased resistance, few studies demonstrate causation for these same mutations. Herein, we examined fitness costs associated with metronidazole and amoxicillin resistance. Isogenic strains bearing literature reported point mutations in the rdxA and pbp1 genes were engineered and tested in in vitro competition assays to assess relative fitness. RESULTS: None of the metronidazole resistance mutations resulted in a fitness cost under the tested conditions. In contrast, amoxicillin-resistant mutant strains demonstrated a defect in competition by 24 hours. This change in fitness was further enhanced by moderate osmotic stress. However, under extreme osmotic stress, the amoxicillin-resistant N562Y PBP1 mutant strain showed enhanced fitness, suggesting that there are some pbp1 mutations that can give a conditional fitness advantage. CONCLUSIONS: Our results demonstrate the role of specific point mutations in rdxA and pbp1 in antibiotic resistance and suggest that amoxicillin-resistant strains of H. pylori show environmentally dictated changes in fitness. These later finding may be responsible for the relatively low rates of amoxicillin resistance seen in the United States.

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

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

Microbial Dysbiosis During Simian Immunodeficiency Virus Infection is Partially Reverted with Combination Anti-retroviral Therapy.

Human immunodeficiency virus (HIV) infection is characterized by a massive loss of CD4 T cells in the gastrointestinal tract (GIT) that is accompanied by changes in the gut microbiome and microbial translocation that contribute to inflammation and chronic immune activation. Though highly active antiretroviral therapy (HAART) has led to better long-term outcomes in HIV infected patients, it has not been as effective at reverting pathogenesis in the GIT. Using the simian immunodeficiency virus (SIV) infection model, we show that combination antiretroviral therapy (c-ART) partially reverted microbial dysbiosis observed during SIV infection. Though the relative abundance of bacteria, their richness or diversity did not significantly differ between infected and treated animals, microbial dysbiosis was evident via multiple beta diversity metrics: Jaccard similarity coefficient, Bray-Curtis similarity coefficient, and Yue & Clayton theta similarity coefficient. Principal coordinates analysis (PCoA) clustered SIV-infected untreated animals away from healthy and treated animals that were clustered closely, indicating that c-ART partially reversed the gut dysbiosis associated with SIV infection. Metastats analysis identified specific operational taxonomic units (OTUs) falling within the Streptococcus, Prevotella, Acinetobacter, Treponema, and Lactobacillus genera that were differentially represented across the three groups. Our results suggest that complete viral suppression with c-ART could potentially revert microbial dysbiosis observed during SIV and HIV infections.

Characterization of Acinetobacter baumannii Copper Resistance Reveals a Role in Virulence.

Acinetobacter baumannii is often highly drug-resistant and causes severe infections in compromised patients. These infections can be life threatening due to limited treatment options. Copper is inherently antimicrobial and increasing evidence indicates that copper containing formulations may serve as non-traditional therapeutics against multidrug-resistant bacteria. We previously reported that A. baumannii is sensitive to high concentrations of copper. To understand A. baumannii copper resistance at the molecular level, herein we identified putative copper resistance components and characterized 21 strains bearing mutations in these genes. Eight of the strains displayed a copper sensitive phenotype (pcoA, pcoB, copB, copA/cueO, copR/cusR, copS/cusS, copC, copD); the putative functions of these proteins include copper transport, oxidation, sequestration, and regulation. Importantly, many of these mutant strains still showed increased sensitivity to copper while in a biofilm. Inductively coupled plasma mass spectrometry revealed that many of these strains had defects in copper mobilization, as the mutant strains accumulated more intracellular copper than the wild-type strain. Given the crucial antimicrobial role of copper-mediated killing employed by the immune system, virulence of these mutant strains was investigated in Galleria mellonella; many of the mutant strains were attenuated. Finally, the cusR and copD strains were also investigated in the murine pneumonia model; both were found to be important for full virulence. Thus, copper possesses antimicrobial activity against multidrug-resistant A. baumannii, and copper sensitivity is further increased when copper homeostasis mechanisms are interrupted. Importantly, these proteins are crucial for full virulence of A. baumannii and may represent novel drug targets.

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.

Gender differences in innate responses and gene expression profiles in memory CD4 T cells are apparent very early during acute simian immunodeficiency virus infection.

Gender differences in Human immunodeficiency virus (HIV) disease progression and comorbidities have been extensively reported. Using the simian immunodeficiency virus (SIV) infected rhesus macaque model, we show that these differences are apparent very early during the course of infection. Though there were no major changes in the proportions of CD4 T cells or its subsets, central memory CD4 T cells from female macaques were found to differentially regulate a significantly larger number of genes at day 4 post-infection (PI) as compared to males. Pathway analysis revealed divergence of both canonical and biological pathways that persisted at day 10 PI. Changes in gene expression profiles were accompanied by a significant increase in plasma levels of pro-inflammatory mediators such as MCP-1/CCL2, I-TAC/CXCL11, and MIF. Though plasma levels of IFNα did not differ between male and female macaques, the expression levels of IFNα subtype-14, 16, IFNß, and IFNω were significantly upregulated in the lymph nodes of female macaques at day 10 PI as compared to male macaques. Our results suggest that the pathogenic sequelae seen during chronic infection may be shaped by gender differences in immune responses induced very early during the course of HIV infection.