Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

Risk factors for community-associated Clostridioides difficile infection in young children.

The majority of paediatric Clostridioides difficile infections (CDI) are community-associated (CA), but few data exist regarding associated risk factors. We conducted a case-control study to evaluate CA-CDI risk factors in young children. Participants were enrolled from eight US sites during October 2014-February 2016. Case-patients were defined as children aged 1-5 years with a positive C. difficile specimen collected as an outpatient or ⩽3 days of hospital admission, who had no healthcare facility admission in the prior 12 weeks and no history of CDI. Each case-patient was matched to one control. Caregivers were interviewed regarding relevant exposures. Multivariable conditional logistic regression was performed. Of 68 pairs, 44.1% were female. More case-patients than controls had a comorbidity (33.3% vs. 12.1%; P = 0.01); recent higher-risk outpatient exposures (34.9% vs. 17.7%; P = 0.03); recent antibiotic use (54.4% vs. 19.4%; P < 0.0001); or recent exposure to a household member with diarrhoea (41.3% vs. 21.5%; P = 0.04). In multivariable analysis, antibiotic exposure in the preceding 12 weeks was significantly associated with CA-CDI (adjusted matched odds ratio, 6.25; 95% CI 2.18-17.96). Improved antibiotic prescribing might reduce CA-CDI in this population. Further evaluation of the potential role of outpatient healthcare and household exposures in C. difficile transmission is needed.

Epidemiology and factors associated with candidaemia following Clostridium difficile infection in adults within metropolitan Atlanta, 2009-2013.

We assessed prevalence of and risk factors for candidaemia following Clostridium difficile infection (CDI) using longitudinal population-based surveillance. Of 13 615 adults with CDI, 113 (0·8%) developed candidaemia in the 120 days following CDI. In a matched case-control analysis, severe CDI and CDI treatment with vancomycin + metronidazole were associated with development of candidaemia following CDI.

Prevalence of blaZ gene types and the inoculum effect with cefazolin among bloodstream isolates of methicillin-susceptible Staphylococcus aureus.

We sought to define the prevalence of blaZ gene types and the inoculum effect to cefazolin among methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. The blaZ gene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type A blaZ strains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

Invasive Methicillin-Resistant Staphylococcus aureus USA500 Strains from the U.S. Emerging Infections Program Constitute Three Geographically Distinct Lineages.

USA500 isolates are clonal complex 8 (CC8) Staphylococcus aureus strains closely related to the prominent community- and hospital-associated USA300 group. Despite being relatively understudied, USA500 strains cause a significant burden of disease and are the third most common methicillin-resistant S. aureus (MRSA) strains identified in the U.S. Emerging Infections Program (EIP) invasive S. aureus surveillance. To better understand the genetic relationships of the strains, we sequenced the genomes of 539 USA500 MRSA isolates from sterile site infections collected through the EIP between 2005 and 2013 in the United States. USA500 isolates fell into three major clades principally separated by their distribution across different U.S. regions. Clade C1 strains, found principally in the Northeast, were associated with multiple IS256 insertion elements in their genomes and higher levels of antibiotic resistance. C2 was associated with Southern states, and E1 was associated with Western states. C1 and C2 strains all shared a frameshift in the gene encoding AdsA surface-attached surface protein. We propose that the term "USA500" should be used for CC8 strains sharing a recent common ancestor with the C1, C2, and E1 strains but not in the USA300 group.IMPORTANCE In this work, we have removed some of the confusion surrounding the use of the name "USA500," placed USA500 strains in the context of the CC8 group, and developed a strategy for assignment to subclades based on genome sequence. Our new phylogeny of USA300/USA500 will be a reference point for understanding the genetic adaptations that have allowed multiple highly virulent clonal strains to emerge from within CC8 over the past 50 years.

Changing characteristics of invasive pneumococcal disease in Metropolitan Atlanta, Georgia, after introduction of a 7-valent pneumococcal conjugate vaccine.

BACKGROUND: The rate of invasive pneumococcal disease (IPD) has decreased among both immunized children and nonimmunized adults since the licensure of a heptavalent pneumococcal conjugate vaccine (PCV7) for use in infants in the United States in 2000. METHODS: Temporal trends in IPD incidence, clinical syndromes, and underlying conditions were analyzed using active laboratory- and population-based surveillance data from the Centers for Disease Control and Prevention-sponsored Georgia Emerging Infections Program for the 20-county Metropolitan Atlanta, Georgia, for the period of July 1997 through June 2004. P values were determined by test for trend. RESULTS: Since 2000, there have been significant decreases in the rates of invasive pneumococcal pneumonia (relative risk [RR], 0.80; P=.002) and meningitis (RR, 0.41; P=.003) in adults and for primary bacteremia, invasive pneumonia, and meningitis in children (RR, 0.16 [P<.001], 0.60 [P=.003], and 0.70 [P=.009], respectively). Among human immunodeficiency virus-infected persons, there were significant decreases in the overall rates of IPD (decrease of 43%; P<.001) and invasive pneumonia (decrease of 44%; P<.001) since 2000-2001, although the rate of IPD increased significantly (increase of 53%; P=.022) among patients with acquired immunodeficiency syndrome. There was a concurrent increase in the proportion of adults aged > or = 40 years with underlying comorbidities. Rates of non-PCV7 serotypes increased 1.61-fold and 1.28-fold from 2000-2001 to 2003-2004 in children and adults (P=.005 for both). CONCLUSIONS: The decreasing incidence of IPD in Atlanta since 2000-2001 was associated with decreases in cases of pneumonia and meningitis in adult and pediatric subjects and in cases of primary bacteremia in children. The burden of serotype-replacement disease remained small. Adults with comorbidities represent a growing proportion of patients with IPD.

Evaluation of effectiveness of the 23-valent pneumococcal capsular polysaccharide vaccine for HIV-infected patients.

BACKGROUND: We conducted a retrospective case-control study to evaluate effectiveness of pneumococcal vaccine against invasive disease among adults with human immunodeficiency virus (HIV) infection in San Francisco, Calif, and Atlanta, Ga. METHODS: Case patients were 18- to 55-year-old subjects with HIV infection who were admitted to selected hospitals in Atlanta or San Francisco from February 1992 to April 1995 from whom Streptococcus pneumoniae was isolated from a normally sterile site. Controls were HIV-infected patients of similar age matched to cases by hospital of admission and CD4 lymphocyte count (<0.20, 0.20-0.499, >/=0.50 x 10(9)/L [<200, 200-499, >/=500 cells/mm(3)]) or clinical stage of acquired immunodeficiency syndrome. Case and control subjects were restricted to persons known to have HIV infection before hospital admission. Analysis used matched univariate and conditional logistic regression. RESULTS: One hundred seventy-six case patients and 327 controls were enrolled. By univariate analysis, persons with pneumococcal disease were more likely to be black, be current smokers, and have close contact with children. Adjusted for these factors and CD4 cell count, pneumococcal vaccine effectiveness was 49% (95% confidence interval [CI], 12%-70%). Adjusting for all variables and key interaction terms, vaccine effectiveness among whites was 76% (95% CI, 35%-91%), whereas effectiveness among blacks was 24% (95% CI, -50% to 61%). Among controls, vaccination was significantly less common among blacks (29% vs 45%; P<.005). CONCLUSIONS: Pneumococcal vaccine demonstrated protection against invasive pneumococcal infections among white but not black HIV-infected adults. Failure to demonstrate effectiveness among blacks may be due to limited power because of low use of the vaccine in this population, immunization at more advanced stages of immunosuppression, or unmeasured factors. These data support current recommendations for use of pneumococcal vaccine in HIV-infected persons and highlight a clear need for strategies to improve vaccine-induced protection.

Electron tomographic structure and protein composition of isolated rat cerebellar, hippocampal and cortical postsynaptic densities.

Electron tomography and immunogold labeling were used to analyze similarities and differences in the morphology and protein composition of postsynaptic densities (PSDs) isolated from adult rat cerebella, hippocampi, and cortices. There were similarities in physical dimensions and gross morphology between cortical, hippocampal and most cerebellar PSDs, although the morphology among cerebellar PSDs could be categorized into three distinct groups. The majority of cerebellar PSDs were composed of dense regions of protein, similar to cortical and hippocampal PSDs, while others were either composed of granular or lattice-like protein regions. Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, ßCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs. The scaffold molecule PSD-95, a major component of cortical PSDs, was found absent in a fraction of cerebellar PSDs and when present was clustered in its distribution. In contrast, immunogold labeling for the proteasome was significantly more abundant in cerebellar and hippocampal PSDs than cortical PSDs. Together, these results indicate that PSDs exhibit remarkable diversity in their composition and morphology, presumably as a reflection of the unique functional demands placed on different synapses.

Carriage of Haemophilus influenzae type b in children after widespread vaccination with conjugate Haemophilus influenzae type b vaccines.

Rates of invasive Haemophilus influenzae type b (Hib) disease in children decreased very rapidly after licensure of Hib conjugate vaccines. A role for a vaccine-related reduction in nasopharyngeal carriage of Hib has been suggested. We studied oropharyngeal carriage of Hib and vaccination rates in a population of 2- to 5-year-old children in metropolitan Atlanta. Among 584 children 75% were vaccinated with an Hib conjugate vaccine, 17% had not been vaccinated and 8% had no vaccination records available. Forty-one percent of the children were colonized with H. influenzae. One child was colonized with Hib. Hib carriage (0.17%; upper 95% confidence interval boundary, 0.97%) was substantially lower than the estimates of Hib carriage from prior studies of children who had not received Hib conjugate vaccines. Our data are consistent with a decline in Hib carriage induced by widespread use of conjugate Hib vaccines, which may have contributed to the decline of Hib disease in United States children.

Group B streptococcal infection in older patients. Spectrum of disease and management strategies.

Group B streptococcal infection has recently been recognised as an important and apparently increasingly common cause of invasive disease in nonpregnant adults. The annual incidence of invasive disease has been estimated at 4.4 per 100,000 nonpregnant adults and is highest among adults over 60 years of age. The most common clinical diagnoses include skin and soft-tissue infections, bacteraemia with no identified source, osteomyelitis, urosepsis and pneumonia. Other important but less common infections include peritonitis, infectious arthritis, meningitis and endocarditis. The majority of adults with group B streptococcal infections have underlying diseases including diabetes mellitus, malignant neoplasms and liver disease. Nosocomial infection and polymicrobial bacteraemia occur in a significant proportion of patients with invasive group B streptococcal disease. Mortality from invasive disease is particularly high in the elderly. For treatment of serious group B streptococcal infections, high doses of benzylpenicillin (penicillin G) are recommended because of the somewhat higher minimal inhibitory concentrations. In addition to parenteral antibiotic therapy surgical management may be required for successful treatment, particularly in the case of soft-tissue or bone infection. Invasive group B streptococcal infection is a major problem in elderly adults and those with chronic diseases, and efforts should be made to identify and treat such infections early. Future approaches may include vaccine prevention of serious group B streptococcal infection in adults at highest risk.

Emerging and reemerging infectious diseases: a multidisciplinary perspective.

Predictions that infectious diseases would be eliminated as a major threat to human health have been shattered by emerging and reemerging infections, among them acquired immunodeficiency syndrome (AIDS), hemorrhagic fevers, marked increases in infections caused by antimicrobial-resistant bacteria, and the resurgence of tuberculosis and malaria. Understanding the dynamics of emerging and reemerging infections is critical to efforts to reduce the morbidity and mortality of such infections, to establish policy related to preparedness for infectious threats, and for decisions on where to use limited resources in the fight against infections. In order to offer a multidisciplinary perspective, 23 infectious disease specialists, epidemiologists, geneticists, microbiologists, and population biologists participated in an open forum at Emory University on emerging and reemerging infectious diseases. As summarized below, the group addressed questions about the definition, the identification, the factors responsible for, and multidisciplinary approaches to emerging and reemerging infections.

Electron cryotomography of postsynaptic densities during development reveals a mechanism of assembly.

Postsynaptic densities (PSDs) are responsible for organizing receptors and signaling proteins that regulate excitatory transmission in the mammalian brain. To better understand the assembly and 3D organization of this synaptic structure, we employed electron cryotomography to visualize general and fine structural details of PSDs isolated from P2, P14, P21 and adult forebrain in the absence of fixatives and stains. PSDs at P2 are a loose mesh of filamentous and globular proteins and during development additional protein complexes are recruited onto the mesh. Quantitative analysis reveals that while the surface area of PSDs is relatively constant, the thickness and protein occupancy of the PSD volume increase dramatically between P14 and adult. One striking morphological feature is the appearance of lipid raft-like structures, first evident in PSDs from 14 day old animals. These detergent-resistant membranes stain for GM1 ganglioside and their terminations can be clearly seen embedded in protein "bowls" within the PSD complex. In total, these results lead to the conclusion that the PSD is assembled by the gradual recruitment and stabilization of proteins within an initial mesh that systematically adds complexity to the structure.

Group B streptococcal disease in nonpregnant adults.

Group B streptococcal (GBS) disease in nonpregnant adults is increasing, particularly in elderly persons and those with significant underlying diseases. Diabetes, neurological impairment, and cirrhosis increase risk for invasive GBS disease. Skin, soft-tissue, and osteoarticular infections, pneumonia, and urosepsis are common presentations. Meningitis and endocarditis are less common but associated with serious morbidity and mortality. Disease is frequently nosocomial and may be related to the placement of an iv catheter. Recurrent infection occurs in 4.3% of survivors. Capsular serotypes Ia, III, and V account for the majority of disease in nonpregnant adults. Although group B streptococci are susceptible to penicillin, minimum inhibitory concentrations are 4-fold to 8-fold higher than for group A streptococci. Resistance to erythromycin and clindamycin is increasing. The role of antibodies in protection against GBS disease in nonpregnant adults is unresolved. However, the immunogenicity of GBS vaccines being developed for prevention of neonatal disease should be assessed for adults who are at risk.

Cloning and sequence analysis of the structural pilin gene of Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius.

We have cloned and sequenced the Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (Hae) pilin gene. The sequence contained a 648-bp open reading frame encoding a mature pilin protein of 191 amino acids with a calculated mass of 20.5 kDa. There was 82% homology between the open reading frames of the BPF strain F3031 and H. influenzae type b (Hib) (strain M43) pilin genes and 71% homology at the amino acid level between the mature pilin proteins. However, areas of diversity were noted throughout the gene. A 17-bp probe corresponding to an area of diversity in the N-terminal region of the BPF-associated gene hybridized with other BPF strains but not with non-BPF Hae or Hib. In summary, the pilin protein of BPF-associated Hae is highly homologous to Hib pilin yet remains structurally distinct.

Pathogenic events during infection of the human nasopharynx with Neisseria meningitidis and Haemophilus influenzae.

The human nasopharynx is the natural habitat and reservoir for Neisseria meningitidis and Haemophilus influenzae type b (Hib). Meningococcal and Hib pathogenesis was studied in human nasopharyngeal tissue in organ culture. Inocula of greater than or equal to 10(6) meningococci or cfu of Hib were required for consistent production of infection in these cultures. By 24 hours meningococci and Hib grew to 10(8)-10(10) cfu/mL in culture supernatants, while 10(4)-10(7) cfu per organ culture were tissue associated. These studies further indicated that nasopharyngeal mucus contains components that specifically bind Hib; that both meningococci and Hib cause cytotoxicity, resulting in breakdown of tight junctions of epithelial cells, sloughing of ciliated cells, and ciliostasis; that pili are the most important components mediating initial attachment of meningococci to non-ciliated epithelial cells of the human nasopharynx; that Hib expresses both pilus and nonpilus adhesions that facilitate attachment to nonciliated cells; and that meningococci and Hib both invade the epithelial surface to reach the submucosa but do so by different routes. Meningococci and Hib have evolved successful, although divergent, mechanisms by which to infect the human nasopharynx.

Antimicrobial binding of a radiolabeled cationic neutrophil granule protein.

A 57,000-dalton protein (CAP57) purified from human polymorphonuclear leukocytes has antimicrobial activity against a number of gram-negative bacteria. We developed a procedure using solid-phase Iodo-gen to radiolabel CAP57 without destroying its antibacterial activity. Iodinated and native CAP57 were electrophoretically identical. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels revealed greater than 95% of the 125I in a single heavy band in the 57,000-molecular-weight region. The quantity of [125I]CAP57 bound to bacterial test strains was directly proportional to the sensitivity to CAP57.

Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein.

Bactericidal activity and binding of a 57,000-dalton cationic antimicrobial neutrophil granule protein (CAP57) are determined by the presence on bacteria of O-antigen polysaccharide chains and the availability of negatively charged groups in the lipid A region, the inner core region, or both regions of lipopolysaccharide. Polymyxin B (PMB)-resistant mutants with well-defined alterations in lipid A structure and charge (pmrA) are also more resistant to CAP57. We used biologically active radioiodinated CAP57 to study the characteristics and kinetics of binding to a sensitive Rb lipopolysaccharide chemotype, Salmonella typhimurium SH9178, and the relatively resistant pmrA mutant strain SH7426. Binding occurred rapidly and was specific and saturable. Because CAP57 appears to be bound in a manner similar to that of PMB, competition binding studies were performed. Excess PMB did compete with CAP57 for binding to SH9178. Nonapeptide, a polycationic derivative of PMB that has lost its hydrophobic portions, demonstrated a marked decrease in ability to compete for binding with CAP57 compared with PMB. This demonstrated the importance of hydrophobic binding in the interaction of CAP57 with the microbial surface. Thus, we have shown that binding of CAP57 to SH9178 is specific, saturable, and similar to binding of PMB. Both hydrophobic and ionic properties of CAP57 appear to be necessary for binding.

Nucleotide sequence analysis of hypervariable junctions of Haemophilus influenzae pilus gene clusters.

Haemophilus influenzae pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili, hifABCDE, are found inserted in genomes either between pmbA and hpt (hif-1) or between purE and pepN (hif-2). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of H. influenzae strains. The junctions of the hif-1 cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast, hif-2 junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes hicA and hicB) with evidence of frequent recombination. Variation at hif-2 junctions seems to be tied to multiple copies of a 23-bp Haemophilus intergenic dyad sequence. The hif-1 cluster appears to have originated in biogroup aegyptius strains from invasion of the hpt-pmbA region by a DNA template containing the hif-2 genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile "pathogenicity island" not associated with a phage, transposon, or insertion element.