A Population-Based Assessment of the Impact of 7- and 13-Valent Pneumococcal Conjugate Vaccines on Macrolide-Resistant Invasive Pneumococcal Disease: Emergence and Decline of Streptococcus pneumoniae Serotype 19A (CC320) With Dual Macrolide Resistance Mechanisms.

BACKGROUND: Macrolide efflux encoded by mef(E)/mel and ribosomal methylation encoded by erm(B) confer most macrolide resistance in Streptococcus pneumoniae. Introduction of the heptavalent pneumococcal conjugate vaccine (PCV7) in 2000 reduced macrolide-resistant invasive pneumococcal disease (MR-IPD) due to PCV7 serotypes (6B, 9V, 14, 19F, and 23F). METHODS: In this study, the impact of PCV7 and PCV13 on MR-IPD was prospectively assessed. A 20-year study of IPD performed in metropolitan Atlanta, Georgia, using active, population-based surveillance formed the basis for this study. Genetic determinants of macrolide resistance were evaluated using established techniques. RESULTS: During the decade of PCV7 use (2000-2009), MR-IPD decreased rapidly until 2002 and subsequently stabilized until the introduction of PCV13 in 2010 when MR-IPD incidence decreased further from 3.71 to 2.45/100000 population. In 2003, serotype 19A CC320 isolates containing both mef(E)/mel and erm(B) were observed and rapidly expanded in 2005-2009, peaking in 2010 (incidence 1.38/100000 population), accounting for 36.1% of MR-IPD and 11.7% of all IPD isolates. Following PCV13 introduction, dual macrolide-resistant IPD decreased 74.1% (incidence 0.32/100000 in 2013). However, other macrolide-resistant serotypes (eg, 15A and 35B) not currently represented in PCV formulations increased modestly. CONCLUSIONS: The selective pressures of widespread macrolide use and PCV7 and PCV13 introductions on S. pneumoniae were associated with changes in macrolide resistance and the molecular basis over time in our population. Durable surveillance and programs that emphasize the judicious use of antibiotics need to continue to be a focus of public health strategies directed at S. pneumoniae.

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

Rapid killing of Acinetobacter baumannii by polymyxins is mediated by a hydroxyl radical death pathway.

Acinetobacter baumannii is an opportunistic pathogen that is a cause of clinically significant nosocomial infections. Increasingly, clinical isolates of A. baumannii are extensively resistant to numerous antibiotics, and the use of polymyxin antibiotics against these infections is often the final treatment option. Historically, the polymyxins have been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism based on data demonstrating that polymyxins induce rapid cell death through hydroxyl radical production. Supporting this notion, we found that inhibition of radical production delays the ability of polymyxins to kill A. baumannii. Notably, we demonstrate that this mechanism of killing occurs in multidrug-resistant clinical isolates of A. baumannii and that this response is not induced in a polymyxin-resistant isolate. This study is the first to demonstrate that polymyxins induce rapid killing of A. baumannii and other Gram-negatives through hydroxyl radical production. This significantly augments our understanding of the mechanism of polymyxin action, which is critical knowledge toward the development of adjunctive therapies, particularly given the increasing necessity for treatment with these antibiotics in the clinical setting.

Colourful parrot feathers resist bacterial degradation.

The brilliant red, orange and yellow colours of parrot feathers are the product of psittacofulvins, which are synthetic pigments known only from parrots. Recent evidence suggests that some pigments in bird feathers function not just as colour generators, but also preserve plumage integrity by increasing the resistance of feather keratin to bacterial degradation. We exposed a variety of colourful parrot feathers to feather-degrading Bacillus licheniformis and found that feathers with red psittacofulvins degraded at about the same rate as those with melanin and more slowly than white feathers, which lack pigments. Blue feathers, in which colour is based on the microstructural arrangement of keratin, air and melanin granules, and green feathers, which combine structural blue with yellow psittacofulvins, degraded at a rate similar to that of red and black feathers. These differences in resistance to bacterial degradation of differently coloured feathers suggest that colour patterns within the Psittaciformes may have evolved to resist bacterial degradation, in addition to their role in communication and camouflage.

Rapid Inactivation of Non-Endospore-Forming Bacterial Pathogens by Heat Stabilization is Compatible with Downstream Next-Generation Sequencing.

Introduction: Heat stabilization treatment preserves the in vivo state of biological samples by rapidly inactivating enzymes that cause degradation of proteins and nucleic acids. Historically, proteomics studies used this technique as an alternative to chemical fixation. More recently, microbiologists discovered that heat stabilization treatment rapidly inactivates pathogens present in tissue samples and preserves deoxyribonucleic acid (DNA) in the tissue. However, these recent studies did not investigate the inactivation of high-density bacterial suspensions and the quality of bacterial DNA. Methods and Results: High-density suspensions of Escherichia coli (>109 cfu/mL) were completely inactivated by heat stabilization treatment using the Denator Stabilizor T1 instrument at 72°C and 95°C for 45 seconds. Using the heat stabilization instrument, a panel of 30 species, 20 Gram-negative and 10 non-endospore-forming Gram-positive species, were fully inactivated by treatment (95°C for 45 seconds). DNA was isolated from bacterial suspensions of Gram-negative bacteria, including E. albertii, E. coli, Shigella dysenteriae, and S. flexneri, following inactivation via heat stabilization treatment and without treatment. DNA isolated following heat stabilization treatment was fully compatible with all downstream molecular applications tested, including next-generation sequencing, pulsed-field gel electrophoresis, multiplex polymerase chain reaction (PCR), and real-time PCR. Conclusions and Discussion: Heat stabilization treatment of Gram-negative and non-endospore-forming Gram-positive pathogens completely inactivates high-density bacterial suspensions. This treatment is compatible with downstream DNA molecular assays, including next-generation sequencing, pulsed-field gel electrophoresis, and PCR. Inactivation by heat stabilization is a rapid process that may increase safety by decreasing risks for laboratory-associated infections and risks associated with transportation of infectious materials.

High-Level Macrolide Resistance Due to the Mega Element [mef(E)/mel] in Streptococcus pneumoniae.

Transferable genetic elements conferring macrolide resistance in Streptococcus pneumoniae can encode the efflux pump and ribosomal protection protein, mef(E)/mel, in an operon of the macrolide efflux genetic assembly (Mega) element- or induce ribosomal methylation through a methyltransferase encoded by erm(B). During the past 30 years, strains that contain Mega or erm(B) or both elements on Tn2010 and other Tn916-like composite mobile genetic elements have emerged and expanded globally. In this study, we identify and define pneumococcal isolates with unusually high-level macrolide resistance (MICs > 16 µg/ml) due to the presence of the Mega element [mef(E)/mel] alone. High-level resistance due to mef(E)/mel was associated with at least two specific genomic insertions of the Mega element, designated Mega-2.IVa and Mega-2.IVc. Genome analyses revealed that these strains do not possess erm(B) or known ribosomal mutations. Deletion of mef(E)/mel in these isolates eliminated macrolide resistance. We also found that Mef(E) and Mel of Tn2010-containing pneumococci were functional but the high-level of macrolide resistance was due to Erm(B). Using in vitro competition experiments in the presence of macrolides, high-level macrolide-resistant S. pneumoniae conferred by either Mega-2.IVa or erm(B), had a growth fitness advantage over the lower-level, mef(E)/mel-mediated macrolide-resistant S. pneumoniae phenotypes. These data indicate the ability of S. pneumoniae to generate high-level macrolide resistance by macrolide efflux/ribosomal protection [Mef(E)/Mel] and that high-level resistance regardless of mechanism provides a fitness advantage in the presence of macrolides.

High-Quality Complete and Draft Genome Sequences for Three Escherichia spp. and Three Shigella spp. Generated with Pacific Biosciences and Illumina Sequencing and Optical Mapping.

Escherichia spp., including E. albertii and E. coli, Shigella dysenteriae, and S. flexneri are causative agents of foodborne disease. We report here reference-level whole-genome sequences of E. albertii (2014C-4356), E. coli (2011C-4315 and 2012C-4431), S. dysenteriae (BU53M1), and S. flexneri (94-3007 and 71-2783).

Macrolide Resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a common commensal and an opportunistic pathogen. Suspected pneumococcal upper respiratory infections and pneumonia are often treated with macrolide antibiotics. Macrolides are bacteriostatic antibiotics and inhibit protein synthesis by binding to the 50S ribosomal subunit. The widespread use of macrolides is associated with increased macrolide resistance in S. pneumoniae, and the treatment of pneumococcal infections with macrolides may be associated with clinical failures. In S. pneumoniae, macrolide resistance is due to ribosomal dimethylation by an enzyme encoded by erm(B), efflux by a two-component efflux pump encoded by mef (E)/mel(msr(D)) and, less commonly, mutations of the ribosomal target site of macrolides. A wide array of genetic elements have emerged that facilitate macrolide resistance in S. pneumoniae; for example erm(B) is found on Tn917, while the mef (E)/mel operon is carried on the 5.4- or 5.5-kb Mega element. The macrolide resistance determinants, erm(B) and mef (E)/mel, are also found on large composite Tn916-like elements most notably Tn6002, Tn2009, and Tn2010. Introductions of 7-valent and 13-valent pneumococcal conjugate vaccines (PCV-7 and PCV-13) have decreased the incidence of macrolide-resistant invasive pneumococcal disease, but serotype replacement and emergence of macrolide resistance remain an important concern.

Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae.

Macrolide resistance in Streptococcus pneumoniae emerged in the U.S. and globally during the early 1990's. The RNA methylase encoded by erm(B) and the macrolide efflux genes mef(E) and mel were identified as the resistance determining factors. These genes are disseminated in the pneumococcus on mobile, often chimeric elements consisting of multiple smaller elements. To better understand the variety of elements encoding macrolide resistance and how they have evolved in the pre- and post-conjugate vaccine eras, the genomes of 121 invasive and ten carriage isolates from Atlanta from 1994 to 2011 were analyzed for mobile elements involved in the dissemination of macrolide resistance. The isolates were selected to provide broad coverage of the genetic variability of antibiotic resistant pneumococci and included 100 invasive isolates resistant to macrolides. Tn916-like elements carrying mef(E) and mel on the Macrolide Genetic Assembly (Mega) and erm(B) on the erm(B) element and Tn917 were integrated into the pneumococcal chromosome backbone and into larger Tn5253-like composite elements. The results reported here include identification of novel insertion sites for Mega and characterization of the insertion sites of Tn916-like elements in the pneumococcal chromosome and in larger composite elements. The data indicate that integration of elements by conjugation was infrequent compared to recombination. Thus, it appears that conjugative mobile elements allow the pneumococcus to acquire DNA from distantly related bacteria, but once integrated into a pneumococcal genome, transformation and recombination is the primary mechanism for transmission of novel DNA throughout the pneumococcal population.

Subversion of host recognition and defense systems by Francisella spp.

Francisella tularensis is a gram-negative intracellular pathogen and the causative agent of the disease tularemia. Inhalation of as few as 10 bacteria is sufficient to cause severe disease, making F. tularensis one of the most highly virulent bacterial pathogens. The initial stage of infection is characterized by the "silent" replication of bacteria in the absence of a significant inflammatory response. Francisella achieves this difficult task using several strategies: (i) strong integrity of the bacterial surface to resist host killing mechanisms and the release of inflammatory bacterial components (pathogen-associated molecular patterns [PAMPs]), (ii) modification of PAMPs to prevent activation of inflammatory pathways, and (iii) active modulation of the host response by escaping the phagosome and directly suppressing inflammatory pathways. We review the specific mechanisms by which Francisella achieves these goals to subvert host defenses and promote pathogenesis, highlighting as-yet-unanswered questions and important areas for future study.