Cassie L. Dohrn

Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth.

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms. A Matter of Life and Breath The CafePress and Zazzle Web sites and most yoga-wear boutiques sport an array of teeshirts, bumper stickers, and water bottles prepared to offer simple advice to those living a harried life: “Just breathe.” Not so simple for a cystic fibrosis (CF) patient. Very early on, physicians recognized that difficulty breathing was the most ominous of the mosaic of symptoms that characterize this syndrome. Indeed, lung disease is the main cause of death in cystic fibrosis patients, but the lack of an animal model that mirrors the CF lung pathology seen in people has slowed translational cystic fibrosis research. Now, Stoltz et al. report findings in cystic fibrosis pigs that survive long enough to develop human-like lung disease. At the heart of this recessive genetic disease is the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride-ion channel. CF-causing mutations in the CFTR gene give rise to an aberrant channel that is defective in its ability to transport ions and water across cell membranes, resulting in a dizzying array of defects in the pancreas, intestines, reproductive system, liver, and lungs. It has been hypothesized that the impaired channel causes cells that line body cavities and passageways to become coated with thick mucus. In such an environment, bacteria thrive, leading to the chronic infections characteristic of this disease. However, the precise mechanisms by which CFTR mutations manifest as the complex phenotypes that constitute CF remain unclear, particularly with respect to the inflamed and infected airways of the CF lung. Despite substantial research efforts, scientists have been unable to achieve two crucial goals,to mold an animal model that mimics human CF lung disease and to pinpoint the trigger of CF lung pathology in pristine airways. Stoltz et al. tackled both of these obstacles by producing genetically modified CF pigs and analyzing their airways from birth to 6 months of age. Their studies revealed a spontaneously arising human-like lung disease that developed over time and had the CF hallmarks: multibacterial infections, inflammation, and mucus buildup. Although the lungs of the newborn CF piglets were not yet inflamed, they were less likely to be sterile and less able to eliminate bacteria that had been introduced into their lungs, relative to wild-type animals. Together, these findings suggest that bacterial infiltration spurs the pattern of lung inflammation and pathogenesis associated with CF. Having a clearer conception of CF lung disease can help clinicians devise preventive treatments that can be initiated early in the lives of CF patients. Such interventions may let CF suffers live and breath more fully. Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). Understanding the pathogenesis of this disease has been hindered, however, by the lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with mutated CFTR genes. We now report that, within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Our finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.

Pneumococcal serotypes before and after introduction of conjugate vaccines, United States, 1999-2011(1.).

Serotyping data for pneumococci causing invasive and noninvasive disease in 2008–2009 and 2010–2011 from >43 US centers were compared with data from preconjugate vaccine (1999–2000) and postconjugate vaccine (2004–2005) periods. Prevalence of 7-valent pneumococcal conjugate vaccine serotypes decreased from 64% of invasive and 50% of noninvasive isolates in 1999–2000 to 3.8% and 4.2%, respectively, in 2010–2011. Increases in serotype 19A stopped after introduction of 13-valent pneumococcal vaccine (PCV13) in 2010. Prevalences of other predominant serotypes included in or related to PCV13 (3, 6C, 7F) also remained similar for 2008–2009 and 2010–2011. The only major serotype that increased from 2008–2009 to 2010–2011 was nonvaccine serotype 35B. These data show that introduction of the 7-valent vaccine has dramatically decreased prevalence of its serotypes and that addition of serotypes in PCV13 could provide coverage of 39% of isolates that continue to cause disease.

The ΔF508 Mutation Causes CFTR Misprocessing and Cystic Fibrosis–Like Disease in Pigs

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs. Four Legs Good, Two Legs Bad In Animal Farm, George Orwell describes a pasture in which the pigs lead an animal revolt, resulting eventually in the porcine dwellers becoming indistinguishable from the human ones against whom they revolted. Scientists similarly wish for pigs to model humans, although as large animal models of human disease, not despotic rulers. Ostedgaard et al. extended this idea to cystic fibrosis (CF), generating pigs that carry the most common human CF mutation, Δ508. CF is a devastating genetic disease characterized by difficulty breathing, progressive disability, persistent infections, and, often, early death. CF is caused by a mutation in the gene that encodes the CF transmembrane conductance regulator (CFTR), which is an anion channel that modulates the components of sweat, digestive juices, and mucus. The most common mutation in CF patients results in an altered version of CFTR, CFTR-Δ508, which is found in 1 of 25 people of Caucasian descent. CF is difficult to study in human patients, and mouse models do not accurately reflect the human disease. Pigs may provide a better model of CF because they have more similar anatomy, biochemistry, physiology, size, and genetics to humans than mice. Thus, the authors generated a pig model of CF with the CFTR-Δ508 mutation. Similar to pigs that completely lack expression of CFTR, the CFTR-Δ508 pigs developed CF symptoms that mimicked those in human patients. In these animals, much of the CFTR-Δ508 protein was misprocessed; specifically, it was retained in the endoplasmic reticulum and rapidly degraded. However, pigs with CFTR-Δ508 retained small amounts of CFTR conductance (~6%), although this level of function was not sufficient to prevent disease. This new model may help to determine which levels of CFTR are sufficient for function and, therefore, guide future therapeutic strategies. After all, all animal models are equal, but some are more equal than others. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTRΔF508/ΔF508 airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3′,5′-monophosphate (cAMP) agonists were less potent at stimulating current in CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/ΔF508 pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

Changing Epidemiology of Antimicrobial-Resistant Streptococcus pneumoniae in the United States, 2004–2005

BACKGROUND The impact of pediatric 7-valent pneumococcal conjugate vaccination (PCV-7) on the population of Streptococcus pneumoniae in the United States was examined by determining the serotypes, antimicrobial resistance profiles, and genetic relatedness of isolates from patients with invasive and noninvasive infections during the 2004-2005 respiratory illness season. METHODS Susceptibility testing, serotyping, and pulsed-field gel electrophoresis analysis were performed on 1647 S. pneumoniae isolates obtained from 41 US medical centers in 2004-2005 as part of a longitudinal antimicrobial resistance surveillance program. The results were compared with surveillance data from earlier periods. RESULTS From the 1999-2000 to the 2004-2005 respiratory illness season, the prevalence of isolates with intermediate penicillin resistance (minimum inhibitory concentration, 0.1-1 microg/mL) increased from 12.7% to 17.9%, prevalence of penicillin-resistant isolates (minimum inhibitory concentration, >or=2 microg/mL) decreased from 21.5% to 14.6%, and prevalence of isolates resistant to erythromycin increased from 25.7% to 29.1% among S. pneumoniae isolates. The prevalence of multidrug resistance among isolates did not change (22.4% in 1999-2000 and 20.0% in 2004-2005). Sixty different serotypes were recognized among the isolates from 2004-2005; predominant serotypes were 19A (14.5%), 3 (11.2%), 6A (7.1%), 19F (7%), and 11A (6%). Serotypes that were included in PCV-7 accounted for 16.3% of isolates; 28.4% of strains isolated had PCV-7-related serotypes, and the remaining 55.3% of isolates had serotypes that were unrelated to PCV-7. The serotype distribution of the penicillin-resistant S. pneumoniae population changed from 1999-2000 to 2004-2005, with an increase in the prevalence of serotype 19A (1.5% to 35.4%) and serotype 35B (1.2% to 12.5%) and a decrease in the prevalence of most PCV-7 serotypes, including 23F (16.1% to 5%), 9V (16.1% to 4.2%), 6B (13.7% to 3.8%), and 14 (18.5% to 2.9%). CONCLUSIONS The penicillin-resistant S. pneumoniae population has changed; most isolates are now closely related to 2 Pneumococcal Molecular Epidemiology Network clones that increased in prevalence from 1999-2000 to 2004-2005 (Taiwan(19F)-14 [14.6% to 36.7%; 60% were serotype 19A] and Utah(35B)-24 [0.9% to 16.3%]).

Changes in Pneumococcal Serotypes and Antimicrobial Resistance after Introduction of the 13-Valent Conjugate Vaccine in the United States

ABSTRACT Ongoing surveillance for Streptococcus pneumoniae is needed to assess the impact of the pneumococcal conjugate vaccine introduced in 2010 (PCV13). Forty-two U.S. centers submitted S. pneumoniae isolates between 1 October 2012 and 31 March 2013. Susceptibility testing was performed by use of a broth dilution method as recommended by the Clinical and Laboratory Standards Institute. Serotyping was performed by multiplex PCR and the Quellung reaction. Multidrug resistance (MDR) was defined as nonsusceptibility to penicillin (PNSP; MIC ≥ 0.12 μg/ml) combined with resistance to ≥2 non-β-lactam antimicrobials. Penicillin-resistant S. pneumoniae (PRSP) was defined as a penicillin MIC of ≥2 μg/ml. For the 1,498 isolates collected during 2012-13, the PRSP and MDR rates were 14.2 and 21.0%, respectively. These percentages were lower than rates obtained in a surveillance study conducted 4 years earlier in 2008-09 (17.0 and 26.6%, respectively). The most common serotypes identified in 2012-13 were 3, 35B, and 19A, each representing 9 to 10% of all isolates. The largest percentage of PNSP in 2012-13 were found in serotypes 35B (24.8%), 19A (23.5%), and 15A (10.3%). Predominant PRSP serotypes were 19A (54.5%), 35B (28.2%), and 19F (7.0%). Major MDR serotypes were 19A (38.5%), 15A (16.9%), 6C (8.3%), and 35B (6.4%). The change in prevalence of PCV13 serotypes (43.4 to 27.1%) was primarily due to a decrease in serotype 19A strains, i.e., 22% of all strains in 2008-09 to 10% of all strains in 2012-13. Among the PNSP subset, serotypes showing a proportional increase were 35B, 15B, and 23B. Among MDR strains, the largest proportional increases were observed in serotypes 35B, 15B, and 23A.

Continued Emergence of USA300 Methicillin-Resistant Staphylococcus aureus in the United States: Results from a Nationwide Surveillance Study

BACKGROUND The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is changing, with USA300 emerging first in community and then in healthcare settings. We performed nationwide surveillance to assess recent trends in the molecular epidemiology of MRSA. METHODS One hundred consecutive unique clinically significant S. aureus isolates were recovered from patients at each of 43 US centers between July 1, 2011, and December 31, 2011. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), staphylococcal protein A gene (spa) and staphylococcal cassette chromosome mec typing, and Panton-Valentine leukocidin detection were performed on all MRSA isolates. RESULTS Of 4,131 isolates collected, 2,093 (51%) were MRSA. Specimen sources of MRSA isolates included wound or abscess (54%), blood (24%), lower respiratory tract (11%), and other sterile site (10%). Thirty percent were isolated more than 48 hours after hospital admission (ie, were associated with nosocomial acquisition of infection). USA300 was the most common PFGE type (1,269 isolates; 61%), overall and in all regions, followed by USA100 (368 isolates; 18%). Among 173 spa types found, the most common were t008 (51%) and t002 (18%); no other spa type accounted for more than 2% of isolates. One strain type (USA300/t008/IV) constituted almost half of all MRSA isolates (1,005 isolates; 48%) and was the most common at all body sites, causing 37% of MRSA bloodstream infections (BSIs) and 38% of nosocomial MRSA infections. Multidrug-resistant phenotypes were found among 34 USA300 isolates (3%) from 18 states. CONCLUSIONS The USA300 PFGE type continues to advance nationwide. A single strain type (USA300/t008/IV) predominates in all regions and infection sites and is now more common than USA100 as a cause of MRSA BSI and nosocomial infections. Although most USA300 retain typical susceptibility profiles, multidrug-resistant phenotypes are emerging.

Activity of Ceftaroline and Epidemiologic Trends in Staphylococcus aureus Isolates Collected from 43 Medical Centers in the United States in 2009

ABSTRACT A Staphylococcus aureus surveillance program was initiated in the United States to examine the in vitro activity of ceftaroline and epidemiologic trends. Susceptibility testing by Clinical and Laboratory Standards Institute broth microdilution was performed on 4,210 clinically significant isolates collected in 2009 from 43 medical centers. All isolates were screened for mecA by PCR and evaluated by pulsed-field gel electrophoresis. Methicillin-resistant S. aureus (MRSA) were analyzed for Panton-Valentine leukocidin (PVL) genes and the staphylococcal cassette chromosome mec (SCCmec) type. All isolates had ceftaroline MICs of ≤2 μg/ml with an MIC50 of 0.5 and an MIC90 of 1 μg/ml. The overall resistance rates, expressed as the percentages of isolates that were intermediate and resistant (or nonsusceptible), were as follows: ceftaroline, 1.0%; clindamycin, 30.2% (17.4% MIC ≥ 4 μg/ml; 12.8% inducible); daptomycin, 0.2%; erythromycin, 65.5%; levofloxacin, 39.9%; linezolid, 0.02%; oxacillin, 53.4%; tetracycline, 4.4%; tigecycline, 0%; trimethoprim-sulfamethoxazole, 1.6%; vancomycin, 0%; and high-level mupirocin, 2.2%. The mecA PCR was positive for 53.4% of the isolates. The ceftaroline MIC90s were 0.25 μg/ml for methicillin-susceptible S. aureus and 1 μg/ml for MRSA. Among the 2,247 MRSA isolates, 51% were USA300 (96.9% PVL positive, 99.7% SCCmec type IV) and 17% were USA100 (93.4% SCCmec type II). The resistance rates for the 1,137 USA300 MRSA isolates were as follows: erythromycin, 90.9%; levofloxacin, 49.1%; clindamycin, 7.6% (6.2% MIC ≥ 4 μg/ml; 1.4% inducible); tetracycline, 3.3%; trimethoprim-sulfamethoxazole, 0.8%; high-level mupirocin, 2.7%; daptomycin, 0.4%; and ceftaroline and linezolid, 0%. USA300 is the dominant clone causing MRSA infections in the United States. Ceftaroline demonstrated potent in vitro activity against recent S. aureus clinical isolates, including MRSA, daptomycin-nonsusceptible, and linezolid-resistant strains.

Detection of Staphylococcus aureus Isolates with Heterogeneous Intermediate-Level Resistance to Vancomycin in the United States

ABSTRACT The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling–area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 μg/ml) or vancomycin-resistant (MIC ≥ 16 μg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 μg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 μg/ml and 0.1% of isolates with vancomycin MICs of 1 μg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.

Accuracy of Phenotypic Methods for Identification of Streptococcus pneumoniae Isolates Included in Surveillance Programs

ABSTRACT Similarities between Streptococcus pneumoniae and viridans group streptococci may result in misidentification of these organisms. In surveillance programs which assess antimicrobial resistance rates among respiratory tract pathogens, such identification errors could lead to overestimates of pneumococcal resistance rates. DNA probe analysis (Gen-Probe, San Diego, CA), the bile solubility test, optochin susceptibility, colony morphology, and the capsular swelling reaction with Omni serum (Staten Serum Institut, Copenhagen, Denmark) were used to characterize 1,733 organisms provisionally identified as S. pneumoniae in a 2004 to 2005 antimicrobial resistance surveillance program. These organisms were obtained in 41 U.S. medical centers. Among these, 1,647 (95%) were determined to be S. pneumoniae by DNA probe. Elimination of those isolates found not to be S. pneumoniae resulted in 1 to 2% decreases in resistance rate estimates with penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. With AccuProbe as a reference standard, the sensitivities and specificities of each phenotypic method for the identification of S. pneumoniae were, respectively, 98.8% and 82.6% for bile solubility, 99.3% and 74.4% for the capsular swelling reaction with Omni serum, and 87.9% and 59.3% for optochin susceptibility. Colony morphology was of limited value, as 391 (23.7%) isolates lacked the typical button or mucoid colony appearance of S. pneumoniae.

Increasing telithromycin resistance among Streptococcus pyogenes in Europe

OBJECTIVES To assess changes in macrolide and ketolide resistance among Streptococcus pyogenes in Europe and to examine the relationship of resistance to antimicrobial usage. METHODS Clinical S. pyogenes isolates were collected from Denmark, Finland, France, Germany, Italy, Netherlands, Norway, Spain, Sweden, UK, Croatia, Hungary, Poland, Slovak Republic and Slovenia during 2002-03 (n = 2165) and 2004-05 (n = 2333). Resistance to telithromycin (MIC > or = 2) and erythromycin (MIC > or = 0.5) was determined by CLSI broth microdilution. Changes in resistance over time and the relationship of resistance to antimicrobial use (European Surveillance of Antimicrobial Consumption data) were assessed. Telithromycin-resistant isolates were characterized by PFGE to determine genetic relatedness and by PCR to detect mef(A), erm(A) and erm(B). RESULTS The erythromycin resistance rate during 2004-05 (11.6%) was similar to 2002-03 (10.4%). The proportion of macrolide-resistant isolates with the constitutive MLS(B) phenotype increased from 29.3% (2002-03) to 45.7% (2004-05). Telithromycin resistance increased from 1.8% in 2002-03 to 5.2% in 2004-05. For Western Europe, associations of telithromycin and erythromycin resistance, respectively, were found with azithromycin use (R2 = 0.52 and 0.60), clarithromycin use (R2 = 0.76 and 0.85) and total macrolide/lincosamide use (R2 = 0.75 and 0.69). For Eastern Europe, associations of antimicrobial use with resistance were not apparent. The 162 telithromycin-resistant isolates comprised 42 PFGE patterns with 68.5% in eight major PFGE groups. The erm(B) gene was detected in 155 of the 162 telithromycin-resistant isolates. CONCLUSIONS Significant increases in telithromycin resistance occurred from 2002-03 to 2004-05 in Europe. Macrolide use appears to be a factor in the emergence of ketolide resistance among S. pyogenes in Western Europe.