Ari Robicsek

Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase

Antimicrobial-modifying resistance enzymes have traditionally been class specific, having coevolved with the antibiotics they inactivate. Fluoroquinolones, antimicrobial agents used extensively in medicine and agriculture, are synthetic and have been considered safe from naturally occurring antimicrobial-modifying enzymes. We describe reduced susceptibility to ciprofloxacin in clinical bacterial isolates conferred by a variant of the gene encoding aminoglycoside acetyltransferase AAC(6′)-Ib. This enzyme reduces the activity of ciprofloxacin by N-acetylation at the amino nitrogen on its piperazinyl substituent. Although approximately 30 variants of this gene have been reported since 1986, the two base-pair changes responsible for the ciprofloxacin modification phenotype are unique to this variant, first reported in 2003 and now widely disseminated. An intense increase in the medical use of ciprofloxacin seems to have been accompanied by a notable development: a single-function resistance enzyme has crossed class boundaries, and is now capable of enzymatically undermining two unrelated antimicrobial agents, one of them fully synthetic.

Plasmid-Mediated Quinolone Resistance: a Multifaceted Threat

SUMMARY Although plasmid-mediated quinolone resistance (PMQR) was thought not to exist before its discovery in 1998, the past decade has seen an explosion of research characterizing this phenomenon. The best-described form of PMQR is determined by the qnr group of genes. These genes, likely originating in aquatic organisms, code for pentapeptide repeat proteins. These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase or topoisomerase IV enzymes from the inhibitory effect of quinolones. Two additional PMQR mechanisms were recently described. aac(6′)-Ib-cr encodes a variant aminoglycoside acetyltransferase with two amino acid alterations allowing it to inactivate ciprofloxacin through the acetylation of its piperazinyl substituent. oqxAB and qepA encode efflux pumps that extrude quinolones. All of these genes determine relatively small increases in the MICs of quinolones, but these changes are sufficient to facilitate the selection of mutants with higher levels of resistance. The contribution of these genes to the emergence of quinolone resistance is being actively investigated. Several factors suggest their importance in this process, including their increasing ubiquity, their association with other resistance elements, and their emergence simultaneous with the expansion of clinical quinolone resistance. Of concern, these genes are not yet being taken into account in resistance screening by clinical microbiology laboratories.

Prevalence in the United States of aac(6′)-Ib-cr Encoding a Ciprofloxacin-Modifying Enzyme

ABSTRACT Among 313 Enterobacteriaceae from the United States with a ciprofloxacin MIC of ≥0.25 μg/ml and reduced susceptibility to ceftazidime, aac(6′)-Ib was present in 50.5% of isolates, and of these, 28% carried the cr variant responsible for low-level ciprofloxacin resistance. aac(6′)-Ib-cr was geographically widespread, stable over time, most common in Escherichia coli, equally prevalent in ciprofloxacin-susceptible and -resistant strains, and not associated with qnr genes.

Universal Surveillance for Methicillin-Resistant Staphylococcus aureus in 3 Affiliated Hospitals

Context Efforts to reduce the frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections have failed until now. Contribution After a baseline year, the authors screened all intensive care unit admissions for MRSA colonization using polymerase chain reaction. In year 3, they screened all hospital admissions. They placed patients who tested positive for MRSA on contact precautions. The prevalence density of MRSA clinical infection was 8.9, 7.4, and 3.9 per 10000 patient-days in years 1, 2, and 3, respectively. Methicillin-sensitive S. aureus infection rates did not change. Caution There was no concomitant, unscreened control group. Implication Screening for MRSA colonization is associated with substantially reduced rates of MRSA clinical infection. The Editors Methicillin-resistant Staphylococcus aureus (MRSA) is now endemic in many U.S. hospitals (1, 2). Colonization with this organism is a risk factor for eventual MRSA clinical infection (3), which is associated with high cost (4) and poor clinical outcomes (5). The burden of health careassociated MRSA disease is high and may be increasing: In their multiregion survey of invasive MRSA disease, investigators for the Centers for Disease Control and Prevention noted substantial increases in both community- and health careassociated infections at several sites when comparing data from 2001 to 2002 with data from 2004 to 2005 (6). Driven by the emerging concern that community-associated MRSA has entered the hospital environment (7), the medical community and the public are seeking to limit the spread of this organism with increasing urgency (8). In the United Kingdom, the Department of Health has instituted mandatory reporting of MRSA infections in hospitals (9), and in the United States, state legislatures are considering (or have passed bills) requiring active surveillance for MRSA (1012). Consumer organizations (13) and the media (14) also seek action. The Healthcare Infection Control Practices Advisory Committee of the Centers for Disease Control and Prevention (15) recently published guidelines recommending expanded surveillance of asymptomatic patients in settings in which multidrug-resistant organisms are poorly controlled with other measures. However, the evidence supporting this practice is limited to surveillance on circumscribed (for example, intensive care only) populations in small, single-center studies at large academic hospitals (1618). Because rates of MRSA infection remained unacceptably high despite conventional interventions, we implemented expanded surveillance at our 3-hospital health care organization in 2 steps. For 12 months, we implemented organization-wide, intensive care unit (ICU)based MRSA surveillance. On 1 August 2005, we initiated the first program (to our knowledge) of universal surveillance of all hospital admissions in the United States. We aimed to determine whether expanded surveillance was associated with changes in the rate of MRSA clinical disease. Methods We measured the utility of expanded surveillance for MRSA by using a 3-period before-and-after design (Figure 1). Period 1 (no active surveillance) was the baseline. In periods 2 and 3, we introduced ICU-based surveillance and universal admission surveillance, respectively. We compared MRSA disease rates during and after hospitalization in the 3 periods. Figure 1. Intervention timeline. ICU = intensive care unit; MRSA = methicillin-resistant Staphylococcus aureus. Outcomes The primary outcome was aggregate hospital-associated MRSA infection rate, defined as the sum of all MRSA bloodstream, respiratory, urinary tract, and surgical site clinical infections occurring more than 48 hours after admission through day 30 after discharge. Secondary outcomes were rates of health careassociated MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, rates of aggregate MRSA infections occurring up to 180 days after discharge, and adherence to MRSA surveillance. We defined adherence as the percentage of admissions (ICU or whole house, depending on the period) in which surveillance testing was done. Study Sites Evanston Northwestern Healthcare, Evanston, Illinois, is a 3-hospital organization with approximately 40000 annual admissions, 75 affiliated off-site offices, 450 staff physicians, and more than 1000 affiliated physicians. Hospital 1 is an academic facility with several residency programs, 476 beds, and a high proportion of surgical patients. Hospital 2 is a primary care teaching hospital with 143 beds that serves a large population of long-term care facility residents (the elderly) relative to the other 2 hospitals. Hospital 3 is a community hospital with 239 beds. The total number of ICU beds was 45 (5.2% of all beds). Surveillance, Isolation, and Decolonization During the baseline year (period 1), routine surveillance for MRSA colonization did not occur. Patients who were MRSA-colonized on the basis of clinical cultures were placed in contact isolation, and decolonization was not attempted. During all periods, contact isolation consisted of a private room or a shared room with another MRSA-colonized patient. Gowns and gloves were required for all room entries, and patient rooms were supplied with dedicated equipment (for example, stethoscopes) for staff use. During period 2, a policy of nasal surveillance for MRSA colonization was enforced for all ICU admissions. Test turnaround time was 2.5 days. Colonized patients were isolated; decolonization therapy was not standard policy. During period 3, a policy of nasal surveillance for MRSA colonization was enforced for all hospitalizations on entry into a ward room (that is, day 1 of admission). A nurse or patient care technician obtained the specimen. Average test turnaround time was 0.67 day. Nursing staff were notified of results by telephone. Adherence was promoted through in-service education for all nursing staff and educational rounds for physicians (19). During this period, we monitored adherence and provided feedback in real time to underperforming nursing units. The infection control department recommended treatment of colonized patients with a 5-day regimen comprising mupirocin calcium, 2% twice daily to the nares, and a chlorhexidine 4% wash or shower every 2 days during period 3. Patients who were discharged before therapy was complete were sent home with prescriptions to complete this regimen. Because we felt that the decision to decolonize should be at the discretion of the physician, we did not monitor adherence to decolonization, nor did we define it as an outcome of the study. However, we had access to pharmacy data for most patients who tested positive for MRSA during the first 12 months of period 3, and we used it to determine adherence to at least 1 chlorhexidine wash and 4 or more doses of mupirocin (a quantity that seems as effective as 10 doses [20]). Patients were not removed from isolation after decolonization therapy unless another test (done at least 7 days after decolonization therapy during the same hospitalization or on repeated hospitalization) was negative for MRSA. Laboratory Methods Polymerase chain reaction tests for S. aureus colonization have better sensitivity than culture-based assays, but they may yield more false-positive results (21). Real-time polymerase chain reaction was used for MRSA detection in periods 2 (22) and 3 (21). Our in-house method and the commercial assay (BD-GeneOhm real-time polymerase chain reaction test, Becton Dickinson, Franklin Lakes, New Jersey) have equal sensitivity (21, 22). For the commercial assay, we modified the package insert protocol for specimen processing to facilitate high-volume testing (21). Data Collection Demographic Characteristics Administrative data were used to determine admission, procedure, and demographic data for all patients hospitalized from 1 August 2003 to 30 April 2007. We used International Classification of Diseases, Ninth Revision, diagnostic and procedure codes to generate comorbidity data according to the method of Elixhauser and coworkers (23) with Healthcare Cost and Utilization Project comorbidity software, version 3.2 (Agency for Healthcare Research and Quality, Rockville, Maryland) (24). Infections To measure the effect of our intervention, we were interested in true clinical disease due to MRSA. Therefore, we reviewed the records of all patients with positive inpatient or outpatient clinical cultures for MRSA from 1 August 2003 to 30 April 2007. Infections were determined as follows: bacteremia = any positive blood culture; bloodstream infection = positive blood culture in the absence of a positive clinical culture from another site; respiratory tract infection = positive respiratory culture, compatible chest radiograph, and decision to treat; urinary tract infection = positive urine culture and either a decision to treat or growth of more than 100000 colony-forming units/mL plus at least 50 leukocytes per high-power field; and surgical site infection = positive culture of a surgical site. These infection types, although not encompassing all MRSA infections at our organization, represent the major body sites affected by culture-demonstrable MRSA disease. Our primary outcome measure was the rate of clinical hospital-associated MRSA infections. Infections occurring more than 2 days after the admission date and within 30 days after discharge were considered hospital-associated. Rates of hospital-associated MRSA and hospital-associated MSSA were expressed as prevalence density of infections, that is, the number of infections per 10000 inpatient-days. Patients were counted once every 30-day period. In a separate analysis of the timing of MRSA infections, disease prevalence (that is, infections per 10000 admissions) was measured during admission and in 6 postdischarge, 30-day time frames. Statistical Analysis Rates of MRSA Infection For 1 hospital-associated infection analysis, we compared infection

qnr Prevalence in Ceftazidime-Resistant Enterobacteriaceae Isolates from the United States

ABSTRACT We screened 313 ceftazidime-resistant Enterobacteriaceae isolates obtained in the United States from 1999 to 2004 for all three known qnr genes. A qnr gene was present in 20% of Klebsiella pneumoniae isolates, 31% of Enterobacter sp. isolates, and 4% of Escherichia coli isolates. qnrA and qnrB occurred with equivalent frequencies and, except for qnrB in enterobacters, were stable over time. qnrS was absent.

qnrB, Another Plasmid-Mediated Gene for Quinolone Resistance

ABSTRACT A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 β-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to ≥95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 μg/ml (6 μM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 β-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.

Detection of Toxigenic Clostridium difficile in Stool Samples by Real-Time Polymerase Chain Reaction for the Diagnosis of C. difficile-Associated Diarrhea

BACKGROUND Clostridium difficile-associated diarrhea (CDAD) is the major cause of health care-associated infectious diarrhea. Current laboratory testing lacks a single assay that is sensitive, specific, and rapid. The purpose of this work was to design and validate a sensitive and specific real-time polymerase chain reaction (PCR) diagnostic test for CDAD. METHODS This observational validation study of a new real-time PCR assay occurred from July 2004 through April 2006 and involved the testing of 1368 stool samples. As the final validation portion of the investigation, 350 inpatients were prospectively interviewed for clinical findings for 365 episodes of diarrheal illness. Test results and clinical criteria were used to assess the performance of 4 assays. RESULTS Using clinical criteria requiring at least 3 loose stools in 1 day as part of the reference standard for a positive test result supporting CDAD, the sensitivity, specificity, and positive and negative predictive values were 73.3%, 97.6%, 73.3%, and 97.6%, respectively, for enzyme immunoassay; 93.3%, 97.4%, 75.7%, and 99.4%, respectively, for real-time PCR; 76.7%, 97.1%, 69.7%, and 97.9%, respectively, for cell culture cytotoxin assay; and 100.0%, 95.9%, 68.2%, and 100.0%, respectively, for anaerobic culture (for toxigenic C. difficile strains). The real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P<.01 to P<.05). CONCLUSIONS With an assay turnaround time of <4 h, real-time PCR is a more sensitive and equally rapid test, compared with enzyme immunoassay, and is a feasible laboratory option to replace enzyme immunoassay for toxigenic C. difficile detection in clinical practice, as well as for use during the development of new therapeutic agents.

Plasmid-Mediated Quinolone Resistance in Non-Typhi Serotypes of Salmonella enterica

BACKGROUND Serious infections with Salmonella species are often treated with fluoroquinolones or extended-spectrum beta-lactams. Increasingly recognized in Enterobacteriaceae, plasmid-mediated quinolone resistance is encoded by qnr genes. Here, we report the presence of qnr variants in human isolates of non-Typhi serotypes of Salmonella enterica (hereafter referred to as non-Typhi Salmonella) from the United States National Antimicrobial Resistance Monitoring System for Enteric Bacteria. METHODS All non-Typhi Salmonella specimens from the United States National Antimicrobial Resistance Monitoring System for Enteric Bacteria collected from 1996 to 2003 with ciprofloxacin minimum inhibitory concentrations > or = 0.06 microg/mL (233 specimens) and a subset with minimum inhibitory concentrations < or = 0.03 microg/mL (102 specimens) were screened for all known qnr genes (A, B, and S) by polymerase chain reaction. For isolates with positive results, qnr and quinolone resistance-determining region sequences were determined. Plasmids containing qnr genes were characterized by conjugation or transformation. RESULTS Conjugative plasmids harboring qnrB variants were detected in 7 Salmonella enterica serotype Berta isolates and 1 Salmonella enterica serotype Mbandaka isolate. The S. Mbandaka plasmid also had an extended-spectrum beta -lactamase. Variants of qnrS on nonconjugative plasmids were detected in isolates of Salmonella enterica serotype Anatum and Salmonella enterica serotype Bovismorbificans. CONCLUSIONS Plasmid-mediated quinolone resistance appears to be widely distributed, though it is still uncommon in non-Typhi Salmonella isolates from the United States, including strains that are quinolone susceptible by the criteria of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). The presence of this gene in non-Typhi Salmonella that causes infection in humans suggests potential for spread through the food supply, which is a public health concern.

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.

Performance of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Test before and during High-Volume Clinical Use

ABSTRACT We evaluated the use of the BD GeneOhm MRSA real-time PCR assay (BD Diagnostics, San Diego, CA) for the detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and an in-house lysis method that was specifically developed to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were 98%, 96%, 77%, and 99.7% with the kit lysate and 98%, 95%, 75%, and 99.7% with the ACP lysate (P, not significant), respectively. The second evaluation was done after implementation of all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed an NPV of 99.9% and a PPV of 73.5% (prevalence, 6%), consistent with our initial findings. The BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When one is dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.