Patrick R. Murray

Topographical and temporal diversity of the human skin microbiome.

The Close and Personal Biome Fortunately, our skin is readily accessible for ecological studies of the microbial communities that influence health and disease states. Grice et al. (p. 1190) present a metagenomic survey of body sites from 10 healthy human individuals sampled over time. Although, altogether 18 phyla were discovered, only a few predominated. The most diverse communities were found on the forearm and the least behind the ear, but between people the microorganisms living behind the knees, in the elbow, and behind the ear were most similar. This finding might have some bearing on the common occurrence of atopic dermatitis in these zones, although no similar relationship was discerned between skin microbial flora and psoriasis. The human skin provides a landscape of dry, damp, and greasy niches for a diversity of symbiotic microorganisms. Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.

Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis

Atopic dermatitis (AD) has long been associated with Staphylococcus aureus skin colonization or infection and is typically managed with regimens that include antimicrobial therapies. However, the role of microbial communities in the pathogenesis of AD is incompletely characterized. To assess the relationship between skin microbiota and disease progression, 16S ribosomal RNA bacterial gene sequencing was performed on DNA obtained directly from serial skin sampling of children with AD. The composition of bacterial communities was analyzed during AD disease states to identify characteristics associated with AD flares and improvement post-treatment. We found that microbial community structures at sites of disease predilection were dramatically different in AD patients compared with controls. Microbial diversity during AD flares was dependent on the presence or absence of recent AD treatments, with even intermittent treatment linked to greater bacterial diversity than no recent treatment. Treatment-associated changes in skin bacterial diversity suggest that AD treatments diversify skin bacteria preceding improvements in disease activity. In AD, the proportion of Staphylococcus sequences, particularly S. aureus, was greater during disease flares than at baseline or post-treatment, and correlated with worsened disease severity. Representation of the skin commensal S. epidermidis also significantly increased during flares. Increases in Streptococcus, Propionibacterium, and Corynebacterium species were observed following therapy. These findings reveal linkages between microbial communities and inflammatory diseases such as AD, and demonstrate that as compared with culture-based studies, higher resolution examination of microbiota associated with human disease provides novel insights into global shifts of bacteria relevant to disease progression and treatment.

Identification of Mycobacteria in Solid-Culture Media by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced into the clinical microbiology laboratory as a rapid and accurate method to identify bacteria and yeasts. In this paper we describe our work on the use of MALDI-TOF MS for the identification of mycobacterial isolates. We developed a protocol for protein extraction from mycobacteria and utilized it to construct a database containing 42 clinically relevant type and reference strains of mycobacteria. The database was used to identify 104 clinical isolates of mycobacteria. All members of the Mycobacterium tuberculosis complex were identified accurately at the complex level but could not be separated at the species level. All other organisms were identified at the species level, with the exception of one strain of M. kansasii (accurately identified but with a low spectral score) and three pairs of closely related strains: M. abscessus and M. massiliense, M. mucogenicum and M. phocaicum, and M. chimaera and M. intracellulare. These pairs of organisms can currently be identified only by multilocus gene sequence analysis. We conclude that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.

Genome-wide recombination drives diversification of epidemic strains of Acinetobacter baumannii

Acinetobacter baumannii is an emerging human pathogen and a significant cause of nosocomial infections among hospital patients worldwide. The enormous increase in multidrug resistance among hospital isolates and the recent emergence of pan-drug–resistant strains underscores the urgency to understand how A. baumannii evolves in hospital environments. To this end, we undertook a genomic study of a polyclonal outbreak of multidrug-resistant A. baumannii at the research-based National Institutes of Health Clinical Center. Comparing the complete genome sequences of the three dominant outbreak strain types enabled us to conclude that, despite all belonging to the same epidemic lineage, the three strains diverged before their arrival at the National Institutes of Health. The simultaneous presence of three divergent strains from this lineage supports its increasing prevalence in international hospitals and suggests an ongoing adaptation to the hospital environment. Further genomic comparisons uncovered that much of the diversification that occurred since the divergence of the three outbreak strains was mediated by homologous recombination across 20% of their genomes. Inspection of recombinant regions revealed that several regions were associated with either the loss or swapping out of genes encoding proteins that are exposed to the cell surface or that synthesize cell-surface molecules. Extending our analysis to a larger set of international clinical isolates revealed a previously unappreciated ability of A. baumannii to vary surface molecules through horizontal gene transfer, with subsequent intraspecies dissemination by homologous recombination. These findings have immediate implications in surveillance, prevention, and treatment of A. baumannii infections.

Penicillin Resistance and Penicillinase Production in Clinical Isolates of Bacteroides melaninogenicus

The minimum inhibitory concentrations (MIC) of penicillin and six other antimicrobials were determined for 50 clinical isolates of Bacteroides melaninogenicus. Agar dilution susceptibilities were performed using supplemented brucella blood agar and the proposed National Committee for Clinical Laboratory Standards standard method for anaerobes; results with the two methods were comparable. A penicillin concentration ≥0.8 μg/ml was needed to inhibit 56% of the isolates, whereas 100% were susceptible to 0.1 μg of clindamycin per ml. All isolates with penicillin MIC values ≥0.8 μg/ml produced β-lactamase using a slide method. A micro-iodometric assay was used to quantitate β-lactamase production in six isolates. The β-lactamase activity of B. melaninogenicus was comparable to that of a Staphylococcus aureus isolate but was not inducible, and the specific amount produced correlated only partially with penicillin MIC values. A clinical review of patients from whom the β-lactamase-producing strains of B. melaninogenicus were isolated did not suggest any increased virulence in these strains or an unexpectedly poor clinical response to appropriate therapy.

Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis

ABSTRACT We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens.

Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus

ABSTRACT Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.

Staphylococcus epidermidis pan-genome sequence analysis reveals diversity of skin commensal and hospital infection-associated isolates

BackgroundWhile Staphylococcus epidermidis is commonly isolated from healthy human skin, it is also the most frequent cause of nosocomial infections on indwelling medical devices. Despite its importance, few genome sequences existed and the most frequent hospital-associated lineage, ST2, had not been fully sequenced.ResultsWe cultivated 71 commensal S. epidermidis isolates from 15 skin sites and compared them with 28 nosocomial isolates from venous catheters and blood cultures. We produced 21 commensal and 9 nosocomial draft genomes, and annotated and compared their gene content, phylogenetic relatedness and biochemical functions. The commensal strains had an open pan-genome with 80% core genes and 20% variable genes. The variable genome was characterized by an overabundance of transposable elements, transcription factors and transporters. Biochemical diversity, as assayed by antibiotic resistance and in vitro biofilm formation, demonstrated the varied phenotypic consequences of this genomic diversity. The nosocomial isolates exhibited both large-scale rearrangements and single-nucleotide variation. We showed that S. epidermidis genomes separate into two phylogenetic groups, one consisting only of commensals. The formate dehydrogenase gene, present only in commensals, is a discriminatory marker between the two groups.ConclusionsCommensal skin S. epidermidis have an open pan-genome and show considerable diversity between isolates, even when derived from a single individual or body site. For ST2, the most common nosocomial lineage, we detect variation between three independent isolates sequenced. Finally, phylogenetic analyses revealed a previously unrecognized group of S. epidermidis strains characterized by reduced virulence and formate dehydrogenase, which we propose as a clinical molecular marker.

A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91phox −/− (X-linked) and p47 phox −/− (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patients original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Kochs postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.

Preventing ventilator-associated pneumonia: does the evidence support the practice?

Ventilator-associated pneumonia (VAP) is among the most common infections in patients requiring endotracheal tubes with mechanical ventilation. Ventilator-associated pneumonia is associated with increased hospital costs, a greater number of days in the intensive care unit, longer duration of mechanical ventilation, and higher mortality. Despite widely accepted recommendations for interventions designed to reduce rates of VAP, few studies have assessed the ability of these interventions to improve patient outcomes. As the understanding of VAP advances and new technologies to reduce VAP become available, studies should directly assess patient outcomes before the health care community implements specific prevention approaches in clinical practice.