Ritu Banerjee

Randomized Trial of Rapid Multiplex Polymerase Chain Reaction–Based Blood Culture Identification and Susceptibility Testing

BACKGROUND The value of rapid, panel-based molecular diagnostics for positive blood culture bottles (BCBs) has not been rigorously assessed. We performed a prospective randomized controlled trial evaluating outcomes associated with rapid multiplex PCR (rmPCR) detection of bacteria, fungi, and resistance genes directly from positive BCBs. METHODS A total of 617 patients with positive BCBs underwent stratified randomization into 3 arms: standard BCB processing (control, n = 207), rmPCR reported with templated comments (rmPCR, n = 198), or rmPCR reported with templated comments and real-time audit and feedback of antimicrobial orders by an antimicrobial stewardship team (rmPCR/AS, n = 212). The primary outcome was antimicrobial therapy duration. Secondary outcomes were time to antimicrobial de-escalation or escalation, length of stay (LOS), mortality, and cost. RESULTS Time from BCB Gram stain to microorganism identification was shorter in the intervention group (1.3 hours) vs control (22.3 hours) (P < .001). Compared to the control group, both intervention groups had decreased broad-spectrum piperacillin-tazobactam (control 56 hours, rmPCR 44 hours, rmPCR/AS 45 hours; P = .01) and increased narrow-spectrum β-lactam (control 42 hours, rmPCR 71 hours, rmPCR/AS 85 hours; P = .04) use, and less treatment of contaminants (control 25%, rmPCR 11%, rmPCR/AS 8%; P = .015). Time from Gram stain to appropriate antimicrobial de-escalation or escalation was shortest in the rmPCR/AS group (de-escalation: rmPCR/AS 21 hours, control 34 hours, rmPCR 38 hours, P < .001; escalation: rmPCR/AS 5 hours, control 24 hours, rmPCR 6 hours, P = .04). Groups did not differ in mortality, LOS, or cost. CONCLUSIONS rmPCR reported with templated comments reduced treatment of contaminants and use of broad-spectrum antimicrobials. Addition of antimicrobial stewardship enhanced antimicrobial de-escalation. CLINICAL TRIALS REGISTRATION NCT01898208.

Escherichia coli Sequence Type 131 Is a Dominant, Antimicrobial- Resistant Clonal Group Associated with Healthcare and Elderly Hosts

OBJECTIVE To determine prevalence, predictors, and outcomes of infection due to Escherichia coli sequence type ST131. DESIGN Retrospective cohort. SETTING All healthcare settings in Olmsted County, Minnesota (eg, community hospital, tertiary care center, long-term care facilities, and ambulatory clinics). PATIENTS Ambulatory and hospitalized children and adults with extraintestinal E. coli isolates. METHODS We analyzed 299 consecutive, nonduplicate extraintestinal E. coli isolates submitted to Olmsted County laboratories in February and March 2011. ST131 was identified using single-nucleotide polymorphism polymerase chain reaction and further evaluated through pulsed-field gel electrophoresis. Associated clinical data were abstracted through medical record review. RESULTS Most isolates were from urine specimens (90%), outpatients (68%), and community-associated infections (61%). ST131 accounted for 27% of isolates overall and for a larger proportion of those isolates resistant to fluoroquinolones (81%), trimethoprim-sulfamethoxazole (42%), gentamicin (79%), and ceftriaxone (50%). The prevalence of ST131 increased with age (accounting for 5% of isolates from those 11-20 years of age, 26% of isolates from those 51-60 years of age, and 50% of isolates from those 91-100 years of age). ST131 accounted for a greater proportion of healthcare-associated isolates (49%) than community-associated isolates (15%) and for fully 76% of E. coli isolates from long-term care facility (LTCF) residents. Multivariable predictors of ST131 carriage included older age, LTCF residence, previous urinary tract infection, high-complexity infection, and previous use of fluoroquinolones, macrolides, and extended-spectrum cephalosporins. With multivariable adjustment, ST131-associated infection outcomes included receipt of more than 1 antibiotic (odds ratio [OR], 2.54 [95% confidence interval (CI), 1.25-5.17]) and persistent or recurrent symptoms (OR, 2.53 [95% CI, 1.08-5.96]). Two globally predominant ST131 pulsotypes accounted for 45% of ST131 isolates. CONCLUSIONS ST131 is a dominant, antimicrobial-resistant clonal group associated with healthcare settings, elderly hosts, and persistent or recurrent symptoms.

A New Clone Sweeps Clean: the Enigmatic Emergence of Escherichia coli Sequence Type 131

ABSTRACT Escherichia coli sequence type 131 (ST131) is an extensively antimicrobial-resistant E. coli clonal group that has spread explosively throughout the world. Recent molecular epidemiologic and whole-genome phylogenetic studies have elucidated the fine clonal structure of ST131, which comprises multiple ST131 subclones with distinctive resistance profiles, including the (nested) H30, H30-R, and H30-Rx subclones. The most prevalent ST131 subclone, H30, arose from a single common fluoroquinolone (FQ)-susceptible ancestor containing allele 30 of fimH (type 1 fimbrial adhesin gene). An early H30 subclone member acquired FQ resistance and launched the rapid expansion of the resulting FQ-resistant subclone, H30-R. Subsequently, a member of H30-R acquired the CTX-M-15 extended-spectrum beta-lactamase and launched the rapid expansion of the CTX-M-15-containing subclone within H30-R, H30-Rx. Clonal expansion clearly is now the dominant mechanism for the rising prevalence of both FQ resistance and CTX-M-15 production in ST131 and in E. coli generally. Reasons for the successful dissemination and expansion of the key ST131 subclones remain undefined but may include increased transmissibility, greater ability to colonize and/or persist in the intestine or urinary tract, enhanced virulence, and more-extensive antimicrobial resistance compared to other E. coli. Here we discuss the epidemiology and molecular phylogeny of ST131 and its key subclones, possible mechanisms for their ecological success, implications of their widespread dissemination, and future research needs.

Extensively Drug-Resistant Tuberculosis in California, 1993–2006

BACKGROUND Extensively drug-resistant (XDR) tuberculosis (TB) is a global public health emergency. We investigated the characteristics and extent of XDR TB in California to inform public health interventions. METHODS XDR TB was defined as TB with resistance to at least isoniazid, rifampin, a fluoroquinolone, and 1 of 3 injectable second-line drugs (amikacin, kanamycin, or capreomycin). Pre-XDR TB was defined as TB with resistance to isoniazid and rifampin and either a fluoroquinolone or second-line injectable agent but not both. We analyzed TB case reports submitted to the state TB registry for the period 1993-2006. Local health departments and the state TB laboratory were queried to ensure complete drug susceptibility reporting. RESULTS Among 424 multidrug-resistant (MDR) TB cases with complete drug susceptibility reporting, 18 (4.2%) were extensively drug resistant, and 77 (18%) were pre-extensively drug resistant. The proportion of pre-XDR TB cases increased over time, from 7% in 1993 to 32% in 2005 (P = .02)). Among XDR TB cases, 83% of cases involved foreign-born patients, and 43% were diagnosed in patients within 6 months after arrival in the United States. Mexico was the most common country of origin. Five cases (29%) of XDR TB were acquired during therapy in California. All patients with XDR TB had pulmonary disease, and most had prolonged infectious periods; the median time for conversion of sputum culture results was 195 days. Among 17 patients with known outcomes, 7 (41.2%) completed therapy, 5 (29.4%) moved, and 5 (29.4%) died. One patient continues to receive treatment. CONCLUSIONS XDR TB and pre-XDR TB cases comprise a substantial fraction of MDR TB cases in California, indicating the need for interventions that improve surveillance, directly observed therapy, and rapid drug susceptibility testing and reporting.

Plasmodium Food Vacuole Plasmepsins Are Activated by Falcipains

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.

Molecular Epidemiology of Escherichia coli Sequence Type 131 and Its H30 and H30-Rx Subclones among Extended-Spectrum-β-Lactamase-Positive and -Negative E. coli Clinical Isolates from the Chicago Region, 2007 to 2010

ABSTRACT We assessed Escherichia coli ST131 and its H30 and H30-Rx subclones for virulence genes, antimicrobial resistance, and extended-spectrum beta-lactamase (ESBL) type. Although both subclones were associated with ESBL production, H30-Rx isolates had higher resistance scores and were associated specifically with CTX-M-15. Three virulence genes (iha, sat, and iutA) were more prevalent among H30 than non-H30 ST131 isolates. Thus, the H30 and H30-Rx subclones are more antimicrobial resistant and have virulence profiles that are distinct from those of non-H30 ST131 isolates.

In Vitro Selection and Characterization of Ceftobiprole-Resistant Methicillin-Resistant Staphylococcus aureus

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to β-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broad-spectrum cephalosporin, ceftobiprole (BPR), binds PBP2a with high affinity and is active against MRSA. We hypothesized that BPR resistance could be mediated by mutations in mecA, the gene encoding PBP2a. We selected BPR-resistant mutants by passage in high-volume broth cultures containing subinhibitory concentrations of BPR. We used strain COLnex (which lacks chromosomal mecA) transformed with pAW8 (a plasmid vector only), pYK20 (a plasmid carrying wild-type mecA), or pYK21 (a plasmid carrying a mutant mecA gene corresponding to five PBP2a mutations). All strains became resistant to BPR by day 9 of passaging, but MICs continued to increase until day 21. MICs increased 256-fold (from 1 to 256 μg/ml) for pAW8, 32-fold (from 4 to 128 μg/ml) for pYK20, and 8-fold (from 16 to 128 μg/ml) for pYK21. Strains carrying wild-type or mutant mecA developed six (pYK20 transformants) or four (pYK21 transformants) new mutations in mecA. The transformation of COLnex with a mecA mutant plasmid conferred BPR resistance, and the loss of mecA converted resistant strains into susceptible ones. Modeling studies predicted that several of the mecA mutations altered BPR binding; other mutations may have mediated resistance by influencing interactions with other proteins. Multiple mecA mutations were associated with BPR resistance in MRSA. BPR resistance also developed in the strain lacking mecA, suggesting a role for chromosomal genes.

The Clonal Distribution and Diversity of Extraintestinal Escherichia coli Isolates Vary According to Patient Characteristics

ABSTRACT The clonal distribution of Escherichia coli across an unselected population in the current era of widespread antimicrobial resistance is incompletely defined. In this study, we used a newly described clonal typing strategy based on sequencing of fumC and fimH (i.e., CH typing) to infer multilocus sequence types (STs) for 299 consecutive, nonduplicate extraintestinal E. coli isolates from all cultures submitted to Olmsted County, MN, laboratories in February and March 2011 and then compared STs with epidemiological data. Forty-seven different STs were identified, most commonly ST131 (27%), ST95 (11%), ST73 (8%), ST127 (6%), and ST69 (5%). Isolates from these five STs comprised two-thirds of health care-associated (HA) isolates but only half of community-associated (CA) isolates. ST131 was represented overwhelmingly (88%) by a single recently expanded H30 subclone, which was the most extensively antimicrobial-resistant subclone overall and was especially predominant in HA infections and among adults >50 years old. In contrast, among patients 11 to 50 years old, ST69, -95, and -73 were more common. Because of the preponderance of the H30 subclone of ST131, ST diversity was lower among HA than CA isolates, and among antimicrobial-resistant than antimicrobial-susceptible isolates, which otherwise had similar ST distributions. In conclusion, in this U.S. Midwest region, the distribution and diversity of STs among extraintestinal E. coli clinical isolates vary by patient age, type of infection, and resistance phenotype. ST131 predominates among young children and the elderly, HA infections, and antimicrobial-resistant isolates, whereas other well-known pathogenic lineages are more common among adolescents and young adults, CA infections, and antimicrobial-susceptible isolates.

A mecA-Negative Strain of Methicillin-Resistant Staphylococcus aureus with High-Level β-Lactam Resistance Contains Mutations in Three Genes

ABSTRACT We previously generated a ceftobiprole-resistant Staphylococcusaureus strain after high inoculum serial passage of a mecA-negative variant of strain COL (R. Banerjee, M. Gretes, L. Basuino, N. Strynadka, and H. F. Chambers, Antimicrob. Agents Chemother. 52:2089-2096, 2008). Genome resequencing of this strain, CRB, revealed that it differs from its parent by five single-nucleotide polymorphisms in three genes, specifically, those encoding PBP4, a low-molecular-weight penicillin-binding protein, GdpP, a predicted signaling protein, and AcrB, a cation multidrug efflux transporter. CRB displayed resistance to a variety of β-lactams but was hypersusceptible to cefoxitin.

Extensively drug-resistant tuberculosis: New strains, new challenges

Extensively drug-resistant (XDR)-TB, defined as TB with resistance to at least isoniazid, rifampin, a fluoroquinolone and either amikacin, kanamycin or capreomycin, is a stark setback for global TB control. Overburdened public-health systems with inadequate resources for case detection and management and high HIV coinfection rates in many regions have contributed to the emergence of XDR-TB. Patients with XDR-TB have poor outcomes, prolonged infectious periods and limited treatment options. To prevent an epidemic of untreatable XDR-TB, improvements in XDR-TB surveillance, increased laboratory capacity for rapid detection of drug-resistant strains, better infection control and the development of new therapeutics are urgently needed.