Unsafe "crossover-use" of chloramphenicol in Uganda: importance of a One Health approach in antimicrobial resistance policy and regulatory action.

Since the introduction of antibiotics into mainstream health care, resistance to these drugs has become a widespread issue that continues to increase worldwide. Policy decisions to mitigate the development of antimicrobial resistance are hampered by the current lack of surveillance data on antibiotic product availability and use in low-income countries. This study collected data on the antibiotics stocked in human (42) and veterinary (21) drug shops in five sub-counties in Luwero district of Uganda. Focus group discussions with drug shop vendors were also employed to explore antibiotic use practices in the community. Focus group participants reported that farmers used human-intended antibiotics for their livestock, and community members obtain animal-intended antibiotics for their own personal human use. Specifically, chloramphenicol products licensed for human use were being administered to Ugandan poultry. Human consumption of chloramphenicol residues through local animal products represents a serious public health concern. By limiting the health sector scope of antimicrobial resistance research to either human or animal antibiotic use, results can falsely inform policy and intervention strategies. Therefore, a One Health approach is required to understand the wider impact of community antibiotic use and improve overall effectiveness of intervention policy and regulatory action

Srv Mediated Dispersal of Streptococcal Biofilms Through SpeB Is Observed in CovRS+ Strains

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms

Detecting imipenem resistance in Acinetobacter baumannii by automated systems (BD Phoenix, Microscan WalkAway, Vitek 2); high error rates with Microscan WalkAway

<p>Abstract</p> <p>Background</p> <p>Increasing reports of carbapenem resistant <it>Acinetobacter baumannii </it>infections are of serious concern. Reliable susceptibility testing results remains a critical issue for the clinical outcome. Automated systems are increasingly used for species identification and susceptibility testing. This study was organized to evaluate the accuracies of three widely used automated susceptibility testing methods for testing the imipenem susceptibilities of <it>A. baumannii </it>isolates, by comparing to the validated test methods.</p> <p>Methods</p> <p>Selected 112 clinical isolates of <it>A. baumanii </it>collected between January 2003 and May 2006 were tested to confirm imipenem susceptibility results. Strains were tested against imipenem by the reference broth microdilution (BMD), disk diffusion (DD), Etest, BD Phoenix, MicroScan WalkAway and Vitek 2 automated systems. Data were analysed by comparing the results from each test method to those produced by the reference BMD test.</p> <p>Results</p> <p>MicroScan performed true identification of all <it>A. baumannii </it>strains while Vitek 2 unidentified one strain, Phoenix unidentified two strains and misidentified two strains. Eighty seven of the strains (78%) were resistant to imipenem by BMD. Etest, Vitek 2 and BD Phoenix produced acceptable error rates when tested against imipenem. Etest showed the best performance with only two minor errors (1.8%). Vitek 2 produced eight minor errors(7.2%). BD Phoenix produced three major errors (2.8%). DD produced two very major errors (1.8%) (slightly higher (0.3%) than the acceptable limit) and three major errors (2.7%). MicroScan showed the worst performance in susceptibility testing with unacceptable error rates; 28 very major (25%) and 50 minor errors (44.6%).</p> <p>Conclusion</p> <p>Reporting errors for <it>A. baumannii </it>against imipenem do exist in susceptibility testing systems. We suggest clinical laboratories using MicroScan system for routine use should consider using a second, independent antimicrobial susceptibility testing method to validate imipenem susceptibility. Etest, whereever available, may be used as an easy method to confirm imipenem susceptibility.</p

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy.</p> <p>Methods</p> <p>Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis.</p> <p>Results</p> <p>Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from <it>in vivo </it>animal models.</p> <p>Conclusion</p> <p>Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine.</p> <p>Trial Registration</p> <p>The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.</p

Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the <it>srv </it>mutant (MGAS5005Δ<it>srv</it>) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the <it>srv </it>mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δ<it>srv </it>to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δ<it>srv </it>was due only to the constitutive production of SpeB or to other changes inherent in the <it>srv </it>mutant strain. To address this question, we constructed a Δ<it>srv</it>Δ<it>speB </it>double mutant through allelic replacement (MGAS5005Δ<it>srv</it>Δ<it>speB</it>) and tested its ability to form biofilms <it>in vitro</it>.</p> <p>Findings</p> <p>Allelic replacement of <it>speB </it>in the <it>srv </it>mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.</p> <p>Conclusions</p> <p>The constitutive production of SpeB by the <it>srv </it>mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of <it>speB</it>/SpeB.</p

Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum