Susan M. Butler-Wu

Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli

BACKGROUND Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Optimization of Periprosthetic Culture for Diagnosis of Propionibacterium acnes Prosthetic Joint Infection

ABSTRACT Propionibacterium acnes is increasingly recognized as an important agent of prosthetic joint infection (PJI). However, the optimum culture conditions for recovery of this organism from PJI specimens have not been determined. By applying a prolonged 28-day culture incubation to all periprosthetic specimens received for bacterial culture from 198 revision arthroplasty procedures, we retrospectively determined that a 13-day culture incubation period is necessary for the recovery of P. acnes from patients with PJI. Incubation beyond this period was associated with increasing recovery of nondiagnostic isolates: 21.7% of P. acnes isolates believed to be clinically unimportant were recovered after 13 days of incubation. Importantly, a diagnosis of P. acnes PJI would have been missed in 29.4% of patients had extended culture incubation been applied only to anaerobic culture media. Although specimens from P. acnes PJIs were more commonly associated with the presence of ≥2 culture media positive for growth, acute inflammation (≥5 neutrophils/high-power field) was observed in only 40% of patients with PJIs that had more than one specimen submitted for bacterial culture. These results support the need for a minimum culture incubation period of 13 days to be applied to both aerobic and anaerobic culture media for all periprosthetic specimens. Optimal recovery of infecting organisms from PJI specimens will be an important component in generating a universal definition for PJI due to indolent agents of infection, such as P. acnes.

Prognostic Factors for Bacterial Cultures Positive for Propionibacterium acnes and Other Organisms in a Large Series of Revision Shoulder Arthroplasties Performed for Stiffness, Pain, or Loosening

BACKGROUND Propionibacterium acnes has been grown on culture in half of the reported cases of chronic infection associated with shoulder arthroplasty. The presence of this organism can be overlooked because its subtle presentation may not suggest the need for culture or because, in contrast to many orthopaedic infections, multiple tissue samples and weeks of culture incubation are often necessary to recover this organism. Surgical decisions regarding implant revision and antibiotic therapy must be made before the results of intraoperative cultures are known. In the present study, we sought clinically relevant prognostic evidence that could help to guide treatment decisions. METHODS We statistically correlated preoperative and intraoperative observations on 193 shoulder arthroplasty revisions that were performed because of pain, loosening, or stiffness with the results of a Propionibacterium acnes-specific culture protocol. Regression models were used to identify factors predictive of a positive culture for Propionibacterium acnes. RESULTS One hundred and eight of the 193 revision arthroplasties were associated with positive cultures; 70% of the positive cultures demonstrated growth of Propionibacterium acnes. The rate of positive cultures per shoulder increased with the number of culture specimens obtained from each shoulder. Fifty-five percent of the positive cultures required observation for more than one week. Male sex, humeral osteolysis, and cloudy fluid were each associated with significant increases of ≥ 600% in the likelihood of obtaining a positive Propionibacterium acnes culture. Humeral loosening, glenoid wear, and membrane formation were associated with significant increases of >300% in the likelihood of obtaining a positive Propionibacterium acnes culture. CONCLUSIONS Preoperative and intraoperative factors can be used to help to predict the risk of a positive culture for Propionibacterium acnes. This evidence is clinically relevant to decisions regarding prosthesis removal or retention and the need for immediate antibiotic therapy at the time of revision shoulder arthroplasty before the culture results become available. LEVEL OF EVIDENCE Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

A Novel Copper-Responsive Regulon in Mycobacterium tuberculosis

In this work we describe the identification of a copper‐inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five‐locus regulon, which includes a gene that encodes the copper‐protective metallothionein MymT, was highly induced in wild‐type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper‐resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR‐regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper‐responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host.

High-Resolution Two-Locus Clonal Typing of Extraintestinal Pathogenic Escherichia coli

ABSTRACT Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach—which we call CH (fum C /fim H ) typing—consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.

Comparison of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Mycobacteria Using Simplified Protein Extraction Protocols

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently been described as a fast and inexpensive method for the identification of mycobacteria. Although mycobacteria require extraction prior to MALDI-TOF MS analysis, previously published protocols have been relatively complex, involving significant hands-on time and materials not often found in the clinical laboratory. In this study, we tested two simplified protein extraction protocols developed at the University of Washington (UW) and by bioMérieux (BMX) for use with two different mass spectrometry platforms (the Bruker MALDI Biotyper and the bioMérieux Vitek MS, respectively). Both extraction protocols included vortexing with silica beads in the presence of ethanol. The commercial Bruker database was also augmented with an in-house database composed of 123 clinical Mycobacterium strains. A total of 198 clinical strains, representing 18 Mycobacterium species, were correctly identified to the species level 94.9% of the time when extracted using the UW protocol and compared to the augmented database. The BMX protocol and Vitek MS system resulted in correct species-level identifications for 94.4% of these strains. In contrast, only 79.3% of the strains were identified to the species level by the nonaugmented Bruker database, although the use of a lower identification score threshold (≥1.7) increased the identification rate to 93.9%, with two misidentifications that were unlikely to be clinically relevant. The two simplified protein extraction protocols described in this study are easy to use for identifying commonly encountered Mycobacterium species.

Identification of substrates of the Mycobacterium tuberculosis proteasome

The putative proteasome‐associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co‐factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co‐A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome‐dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.

Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains

Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.

Origin of Propionibacterium in Surgical Wounds and Evidence-Based Approach for Culturing Propionibacterium from Surgical Sites

BACKGROUND To explore the origin of Propionibacterium in surgical wounds and to suggest an optimized strategy for culturing this organism at the time of revision surgery, we studied the presence of this organism on the skin and in the surgical wounds of patients who underwent revision arthroplasty for reasons other than apparent infection. METHODS Specimens were cultured in broth and on aerobic and anaerobic media. The presence and degree of positivity of Propionibacterium cultures were correlated with sex. The results of dermal and deep cultures were correlated. Times to positivity and the yields of each media type and specimen source were investigated. RESULTS Propionibacterium grew in twenty-three of thirty cultures of specimens obtained preoperatively from the unprepared epidermis over the area where a skin incision was going to be made for a shoulder arthroplasty; males had a greater average degree of positivity than females (p < 0.002). Twelve of twenty-one male subjects and zero of twenty female subjects who had cultures of dermal specimens obtained during revision shoulder arthroplasty had positive findings for Propionibacterium (p = 0.0001). Twelve of twenty male subjects and only one of twenty female subjects had positive deep cultures (p = 0.0004). The positivity of dermal cultures for Propionibacterium was significantly associated with the positivity of deep cultures for this organism (p = 0.0001). If Propionibacterium was present in deep tissues, it was likely that it would be recovered by culture if four different specimens were obtained and cultured for a minimum of seventeen days on three different media: aerobic, anaerobic, and broth. CONCLUSIONS Because the surgical incision of dermal sebaceous glands may be a source of Propionibacterium in deep wounds, strategies for minimizing the risk of Propionibacterium infections may need to be directed at minimizing the contamination of surgical wounds from these bacteria residing in rather than on the skin. Obtaining at least four specimens, observing them for seventeen days, and using three types of culture media optimize the recovery of Propionibacterium at the time of revision surgery.

MALDI-TOF Mass Spectrometry for Microorganism Identification

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, reliable, and high-throughput diagnostic tool for the identification of microorganisms. The technology is unique in clinical microbiology, allowing laboratories to definitively identify bacterial and fungal isolates within minutes. The rapid turnaround time and minimal cost for consumables per specimen compared with conventional identification methods have resulted in MALDI-TOF MS being increasingly used in clinical laboratories worldwide. This article summarizes the current literature on MALDI-TOF MS for microbial identification and provides a preview of the methods potential future applications in clinical microbiology.