Amy E. Kirby

Synergistic action of gentamicin and bacteriophage in a continuous culture population of Staphylococcus aureus.

With the increasing frequency of antibiotic resistance and the decreasing frequency of new antibiotics entering the market, interest has returned to developing bacteriophage as a therapeutic agent. Acceptance of phage therapy, however, is limited by the unknown pharmacodynamics of a replicating agent, as well as the potential for the evolution of resistant bacteria. One way to overcome some of these limitations is to incorporate phage and antibiotics into a dual therapy regimen; however, this increases the complexity of the pharmacodynamics. The aim of this study is to develop an experimental system to evaluate the pharmacodynamics of dual phage-drug therapy. A continuous culture system for Staphylococcus aureus is used to simulate the pharmacokinetics of periodic antibiotic dosing alone and in combination with lytic phage. A computer model representation of the system allows further evaluation of the conditions governing the observed pharmacodynamics. The results of this experimental/modeling approach suggest that dual therapy can be more efficacious than single therapies, particularly if there is an overlap in the physiological pathways targeted by the individual agents. In this case, treatment with gentamicin induces a population of cells with a strong aggregation phenotype. These aggregators also have an increased ability to form biofilm, which is a well-known, non-genetic mechanism of drug resistance. However, the aggregators are also more susceptible than the parental strain to the action of the phage. Thus, dual treatment with gentamicin and phage resulted in lower final cell densities than either treatment alone. Unlike in the phage-only treatment, phage-resistant isolates were not detected in the dual treatment.

Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

ABSTRACT Efficient utilization of heme as an iron (Fe) source byBordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized thatrhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuIwas correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal tobhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced intoEscherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. colidemonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of therhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

The Relative Contributions of Physical Structure and Cell Density to the Antibiotic Susceptibility of Bacteria in Biofilms

ABSTRACT For many bacterial infections, noninherited mechanisms of resistance are responsible for extending the term of treatment and in some cases precluding its success. Among the most important of these noninherited mechanisms of resistance is the ability of bacteria to form biofilms. There is compelling evidence that bacteria within biofilms are more refractory to antibiotics than are planktonic cells. Not so clear, however, is the extent to which this resistance can be attributed to the structure of biofilms rather than the physiology and density of bacteria within them. To explore the contribution of the structure of biofilms to resistance in a quantitative way, we developed an assay that compares the antibiotic sensitivity of bacteria in biofilms to cells mechanically released from these structures. Our method, which we apply to Escherichia coli and Staphylococcus aureus each with antibiotics of five classes, controls for the density and physiological state of the treated bacteria. For most of the antibiotics tested, the bacteria in biofilms were no more resistant than the corresponding populations of planktonic cells of similar density. Our results, however, suggest that killing by gentamicin, streptomycin, and colistin is profoundly inhibited by the structure of biofilms; these drugs are substantially more effective in killing bacteria released from biofilms than cells within these structures.

Disease course and viral shedding in experimental Norwalk virus and Snow Mountain virus infection.

Norovirus is the most common cause of acute infectious gastroenteritis, causing approximately 21 million cases annually in the USA. The virus is highly contagious and resistant to decontamination, making outbreaks difficult to control. To facilitate the development of better control methods, this study characterized the viral shedding patterns in stools from subjects experimentally infected with genogroup I or II norovirus. Viral stool titers were determined by quantitative real‐time RT‐PCR for all stools produced in the first 7 days post‐challenge and representative stools through day 35 post‐challenge. The shedding titers and disease course were analyzed with respect to virus type, illness, and subject demographics. Infection with GII.2 Snow Mountain (SMV) resulted in more symptoms and a higher frequency of painful symptoms compared to GI.1 Norwalk (NV) infection. However, NV infection produced stool viral titers approximately 2 logs higher than those seen in SMV infections. Both NV and SMV were shed in stools for up to 3 weeks after the resolution of symptoms, but long shedding durations were more common in NV infections. For each challenge virus, shedding titers and patterns were not correlated with subject demographics or clinical course. This is the first study to report shedding dynamics in experimental GII norovirus infection. J. Med. Virol. 86:2055–2064, 2014.

Laboratory evidence of norwalk virus contamination on the hands of infected individuals.

ABSTRACT Human norovirus (NoV) outbreak investigations suggest that the hands of infected individuals play an important role in NoV transmission. However, there is no experimental evidence documenting the likelihood and degree of NoV contamination on hands. As part of a clinical trial designed to evaluate the efficacy of high-pressure processing for Norwalk virus (NV) inactivation in oysters, 159 hand rinse samples were collected from 6 infected and 6 uninfected subjects. NV was concentrated from the samples by polyethylene glycol precipitation, followed by RNA extraction using an automated guanidinium isothiocyanate-silica method. NV RNA was detected and quantified using multiple NV-specific reverse transcription-quantitative PCR (RT-qPCR) assays. A total of 25.4% (18/71) of the hand rinse samples collected from 6 infected volunteers were presumptively positive for NV, with an average of 3.86 log10 genomic equivalent copies (GEC) per hand. Dot blot hybridization of PCR products obtained using a different primer set, and DNA sequencing of selected amplicons, provided further confirmation of the presence of NV in the hand rinses. NV contamination was also detected in two hand rinse samples obtained from one uninfected subject. These findings provide definitive evidence of NV contamination on the hands of infected subjects observed under controlled clinical research conditions. Such data support the need for better hand hygiene strategies to prevent NoV transmission.

Vomiting as a Symptom and Transmission Risk in Norovirus Illness: Evidence from Human Challenge Studies

Background In the US, noroviruses are estimated to cause 21 million cases annually with economic losses reaching

RhuR, an Extracytoplasmic Function Sigma Factor Activator, Is Essential for Heme-Dependent Expression of the Outer Membrane Heme and Hemoprotein Receptor of Bordetella avium

2 billion. Outbreak investigations frequently implicate vomiting as a major transmission risk. However, little is known about the characteristics of vomiting as a symptom or the amount of virus present in emesis. Methodology and Principal Findings Emesis samples and symptomology data were obtained from previous norovirus human challenge studies with GI.1 Norwalk virus, GII.2 Snow Mountain virus, and a pilot study with GII.1 Hawaii virus. Viral titers in emesis were determined using strain-specific quantitative RT-PCR. In all four studies, vomiting was common with 40–100% of infected subjects vomiting at least once. However, only 45% of subjects with vomiting also had diarrhea. Most of the emesis samples had detectable virus and the mean viral titers were 8.0 x 105 and 3.9 x 104 genomic equivalent copies (GEC)/ml for GI and GII viruses, respectively (p = 0.02). Sample pH was correlated with GII.2 Snow Mountain virus detection. Conclusions and Significance Half of all subjects with symptomatic infection experienced vomiting and the average subject shed 1.7 x 108 GEC in emesis. Unlike shedding through stool, vomiting is more likely to result in significant environmental contamination, leading to transmission through fomites and airborne droplets. This quantitative data will be critical for risk assessment studies to further understand norovirus transmission and develop effective control measures. The correlation between sample pH and virus detection is consistent with a single site of virus replication in the small intestine and stomach contents becoming contaminated by intestinal reflux. Additionally, the frequency of vomiting without concurrent diarrhea suggests that epidemiology studies that enroll subjects based on the presence of diarrhea may be significantly underestimating the true burden of norovirus disease.

Molecular Detection of Legionella spp. and their associations with Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in a drinking water distribution system.

ABSTRACT Genes involved in iron (Fe) acquisition often are regulated in response to the local availability of Fe. In many bacteria, Fe-dependent responsiveness is mediated by Fur, a global Fe-dependent transcriptional repressor. Tighter regulatory control of Fur-responsive genes is afforded by incorporating additional regulators into Fur-dependent regulatory cascades. RhuI, a Fur-dependent extracytoplasmic function sigma factor of Bordetella avium, in response to the dual stimulation of Fe starvation and the presence of heme (or hemoproteins), regulates PbhuR, a heme-responsive promoter which directs expression of the bhuRSTUV heme utilization operon. While BhuR, the outer membrane heme receptor, and RhuI have been shown to be indispensable for heme-dependent activation of PbhuR, collateral components of the regulatory cascade have not been described. In this investigation, RhuR, an integral cytoplasmic membrane protein with homology to anti-sigma factors, is shown to be an essential activator of PbhuR expression. The functional domain of RhuR required for heme-dependent activation of PbhuR expression was mapped to the N-terminal 97 amino acids of the protein by use of a chimeric RhuR-BlaM fusion. Expression of the chimera in a rhuR mutant rendered PbhuR constitutive, thereby decoupling the promoter from heme dependency. Growth studies confirmed that B. avium requires RhuR for optimal utilization of hemoglobin, but not hemin, as a sole source of nutrient Fe. These data imply that B. avium expresses, in addition to the BhuR heme/hemoprotein utilization system, an alternative RhuR-independent heme utilization mechanism. A model is proposed in which RhuR is the functional bridge between BhuR and RhuI in a heme-dependent regulatory cascade.

The population and evolutionary dynamics of Vibrio cholerae and its bacteriophage: conditions for maintaining phage-limited communities.

This study investigated waterborne opportunistic pathogens (OPs) including potential hosts, and evaluated the use of Legionella spp. for indicating microbial water quality for OPs within a full‐scale operating drinking water distribution system (DWDS).

Transcriptional Control of the rhuIR-bhuRSTUV Heme Acquisition Locus in Bordetella avium

Although bacteriophage have been reported to be the most abundant organisms on earth, little is known about their contribution to the ecology of natural communities of their host bacteria. Most importantly, what role do these viral parasitoids play in regulating the densities of bacterial populations? To address this question, we use experimental communities of Vibrio cholerae and its phage in continuous culture, and we use mathematical models to explore the population dynamic and evolutionary conditions under which phage, rather than resources, will limit the densities of these bacteria. The results of our experiments indicate that single species of bacterial viruses cannot maintain the density of V. cholerae populations at levels much lower than that anticipated on the basis of resources alone. On the other hand, as few as two species of phage can maintain these bacteria at densities more than two orders of magnitude lower than the densities of the corresponding phage-free controls for extensive periods. Using mathematical models and short-term experiments, we explore the population dynamic processes responsible for these results. We discuss the implications of this experimental and theoretical study for the population and evolutionary dynamics of natural populations of bacteria and phage.