A total of 1,531 recent clinical isolates of Streptococcus pneumoniae were collected from 33 medical centers nationwide during the winter of 1999-2000 and characterized at a central laboratory. Of these isolates, 34.2% were penicillin nonsusceptible (MIC > 0.12 g/ml) and 21.5% were high-level resistant (MIC > 2 g/ml). MICs to all beta-lactam antimicrobials increased as penicillin MICs increased. Resistance rates among non-beta-lactam agents were the following: macrolides, 25.2 to 25.7%; clindamycin, 8.9%; tetracycline, 16.3%; chloramphenicol, 8.3%; and trimethoprim-sulfamethoxazole (TMP-SMX), 30.3%. Resistance to non-betalactam agents was higher among penicillin-resistant strains than penicillin-susceptible strains; 22.4% of S. pneumoniae were multiresistant. Resistance to vancomycin and quinupristin-dalfopristin was not detected. Resistance to rifampin was 0.1%. Testing of seven fluoroquinolones resulted in the following rank order of in vitro activity: gemifloxacin > sitafloxacin > moxifloxacin > gatifloxacin > levofloxacin ‫؍‬ ciprofloxacin > ofloxacin. For 1.4% of strains, ciprofloxacin MICs were >4 g/ml. The MIC 90 s (MICs at which 90% of isolates were inhibited) of two ketolides were 0.06 g/ml (ABT773) and 0.12 g/ml (telithromycin). The MIC 90 of linezolid was 2 g/ml. Overall, antimicrobial resistance was highest among middle ear fluid and sinus isolates of S. pneumoniae; lowest resistance rates were noted with isolates from cerebrospinal fluid and blood. Resistant isolates were most often recovered from children 0 to 5 years of age and from patients in the southeastern United States. This study represents a continuation of two previous national studies, one in 1994-1995 and the other in 1997-1998. Resistance rates with S. pneumoniae have increased markedly in the United States during the past 5 years. Increases in resistance from 1994-1995 to 1999-2000 for selected antimicrobial agents were as follows: penicillin, 10.6%; erythromycin, 16.1%; tetracycline, 9.0%; TMP-SMX, 9.1%; and chloramphenicol, 4.0%, the increase in multiresistance was 13.3%. Despite awareness and prevention efforts, antimicrobial resistance with S. pneumoniae continues to increase in the United States.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H+ secretion by the nongastric H+/K+ adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H+; consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.

Nonhealing diabetic foot ulcers (DFUs) are a common and costly complication of diabetes. Microbial burden, or “bioburden,” is believed to underlie delayed healing, although little is known of those clinical factors that may influence microbial load, diversity, and/or pathogenicity. We profiled the microbiomes of neuropathic nonischemic DFUs without clinical evidence of infection in 52 individuals using high-throughput sequencing of the bacterial 16S ribosomal RNA gene. Comparatively, wound cultures, the standard diagnostic in the clinic, vastly underrepresent microbial load, microbial diversity, and the presence of potential pathogens. DFU microbiomes were heterogeneous, even in our tightly restricted study population, but partitioned into three clusters distinguished primarily by dominant bacteria and diversity. Ulcer depth was associated with ulcer cluster, positively correlated with abundance of anaerobic bacteria, and negatively correlated with abundance of Staphylococcus. Ulcer duration was positively correlated with bacterial diversity, species richness, and relative abundance of Proteobacteria, but was negatively correlated with relative abundance of Staphylococcus. Finally, poor glycemic control was associated with ulcer cluster, with poorest median glycemic control concentrating to Staphylococcus-rich and Streptococcus-rich ulcer clusters. Analyses of microbial community membership and structure may provide the most useful metrics in prospective studies to delineate problematic bioburden from benign colonization that can then be used to drive clinical treatment.

Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The “mycobiome” was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes.

It appears that, as fluoroquinolone resistance emerges among S. pneumoniae in the United States, resistance to other antimicrobial classes is becoming less common.