Brian M. Peters

Chromogenic media for MRSA diagnostics.

For the past decade, a number of chromogenic media for methicillin-resistance Staphylococcus aureus (MRSA) detection have been developed and applied, including Oxoid Brilliance™ MRSA, CHROMagar™ MRSA, BBL™ CHROMagar™ MRSA, MRSASelect and chromID MRSA. The advantages of these chromogenic media offers direct detection of visible staphylococcal colonies, coupled with the use of chromogenic enzymatic substrates that can be hydrolyzed by S. aureus to confirm species or strain identification. BBL™ CHROMagar™ MRSA and MRSASelect are designed for detection of nasal colonization by MRSA, while CHROMagar™ MRSA, Oxoid Brilliance™ MRSA and chromID MRSA are readily applied in bacterial screening. This review summarizes the characteristics, principles and capacities of these selective media, and focuses on comparison of different chromogenic media.

Draft genome sequence and annotation of Lactobacillus acetotolerans BM-LA14527, a beer-spoilage bacteria

Lactobacillus acetotolerans is a hard-to-culture beer-spoilage bacterium capable of entering into the viable putative nonculturable (VPNC) state. As part of an initial strategy to investigate the phenotypic behavior of L. acetotolerans, draft genome sequencing was performed. Results demonstrated a total of 1824 predicted annotated genes, with several potential VPNC- and beer-spoilage-associated genes identified. Importantly, this is the first genome sequence of L. acetotolerans as beer-spoilage bacteria and it may aid in further analysis of L. acetotolerans and other beer-spoilage bacteria, with direct implications for food safety control in the beer brewing industry.

The viable but nonculturable state induction and genomic analyses of Lactobacillus casei BM-LC14617, a beer-spoilage bacterium

This study aimed to investigate the viable but nonculturable (VBNC) state and genomic features of a beer‐spoilage strain, Lactobacillus caseiBM‐LC14617. Induction on the VBNC state of L. casei strain BM‐LC14617 was conducted by both low‐temperature storage and continuous passage in beer, and formation of VBNC state was detected after 196 ± 3.3 days and 32 ± 1.6 subcultures, respectively. Resuscitation of VBNC cells was successfully induced by addition of catalase, and culturable, VBNC, and resuscitated cells shared similar beer‐spoilage capability. Whole genome sequencing was performed, and out of a total of 3,964 predicted genes, several potential VBNC and beer‐spoilage‐associated genes were identified. L. casei is capable of entering into and resuscitating from the VBNC state and possesses beer‐spoilage capability. The genomic characterization yield insightful elucidation of VBNC state for L. casei. This study represents the first evidence on VBNC state induction of L. casei and beer‐spoilage capability of VBNC and resuscitated cells. Also, this is the first genomic characterization of L. casei as a beer‐spoilage bacterium. The current study may aid in further study on L. casei and other beer‐spoilage bacteria, and guide the prevention and control of beer spoilage.

Candida–Bacteria Interactions: Their Impact on Human Disease

Candida species are the most common infectious fungal species in humans; out of the approximately 150 known species, Candida albicans is the leading pathogenic species, largely affecting immunocompromised individuals. Apart from its role as the primary etiology for various types of candidiasis, C. albicans is known to contribute to polymicrobial infections. Polymicrobial interactions, particularly between C. albicans and bacterial species, have gained recent interest in which polymicrobial biofilm virulence mechanisms have been studied including adhesion, invasion, quorum sensing, and development of antimicrobial resistance. These trans-kingdom interactions, either synergistic or antagonistic, may help modulate the virulence and pathogenicity of both Candida and bacteria while uniquely impacting the pathogen-host immune response. As antibiotic and antifungal resistance increases, there is a great need to explore the intermicrobial cross-talk with a focus on the treatment of Candida-associated polymicrobial infections. This article explores the current literature on the interactions between Candida and clinically important bacteria and evaluates these interactions in the context of pathogenesis, diagnosis, and disease management.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

ABSTRACT Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.

Complete sequence of pBM413, a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from pseudomonas aeruginosa

This study aimed to characterise a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from Pseudomonas aeruginosa strain Guangzhou-Pae617 isolated from a patient hospitalised in Guangzhou, China, in 2012. The plasmid pBM413 has a length of 423 017 bp and an average G + C content of 56.41%. A qnrVC6 gene flanked by two copies of insertion sequence (IS) elements ISCR1, a multiresistance class 1 integron In786 containing aacA4-blaIMP-45-blaOXA-1-catB3 cassettes, an armA gene, and an aphA7 gene flanked by two copies of IS26 were identified. To our knowledge, this is the first identification of a qnrVC6 gene in P. aeruginosa.

Polymicrobial Biofilm Studies: From Basic Science to Biofilm Control.

Microbes rarely exist as single species planktonic forms as they have been commonly studied in the laboratory. Instead, the vast majority exists as part of complex polymicrobial biofilm communities attached to host and environmental surfaces. The oral cavity represents one of the most diverse and well-studied polymicrobial consortia. Despite a burgeoning field of mechanistic biofilm research within the past decades, our understanding of interactions that occur between microbial members within oral biofilms is still limited. Thus, the primary objective of this review is to focus on polymicrobial biofilm formation, microbial interactions and signaling events that mediate oral biofilm development, consequences of oral hygiene on both local and systemic disease, and potential therapeutic strategies to limit oral dysbiosis.

In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays. ABSTRACT Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

An azole-Tolerant endosomal trafficking mutant of candida albicans is susceptible to azole treatment in a mouse model of vaginal candidiasis

ABSTRACT We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity

ABSTRACT Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicans erg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance. IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted. IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.