Jennifer Adams

Analysis of Antibiotic Resistance Genes in Multidrug-Resistant Acinetobacter sp. Isolates from Military and Civilian Patients Treated at the Walter Reed Army Medical Center

ABSTRACT Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR) Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of blaOXA-58-like and blaPER-like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a “snapshot” of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections.

Tigecycline efflux as a mechanism for nonsusceptibility in acinetobacter baumannii

ABSTRACT Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 μg/ml to 24 μg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter.

Acute Otitis Media Due to Penicillin-Nonsusceptible Streptococcus pneumoniae Before and After the Introduction of the Pneumococcal Conjugate Vaccine

BACKGROUND The impact of the 7-valent pneumococcal conjugate vaccine (PCV7 [Prevnar]) on penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) recovered from children with acute otitis media (AOM) is unclear. METHODS At 5 hospitals, 505 pneumococcal isolates were collected from children with AOM between 1 January 1999 and 31 December 2002. Molecular subtyping was performed on 158 isolates. RESULTS Overall, the percentage of AOM cases due to non-PCV7 serogroups (including serotype 3) increased over time (from 12% in 1999 to 32% in 2002; P < .01) and according to the number of PCV7 doses received (18% [< or = 1 dose] vs. 35% [2-4 doses]; P < .01). The percentage of cases due to vaccine-related serotypes (including serotype 19A) increased according to the number of PCV7 doses received (10% [< or = 1 dose] vs. 19% [2-4 doses]; P = .05) but not over time, whereas the percentage of cases due to serotype 19F remained unchanged both over time and according to the number of PCV7 doses received. The frequency of penicillin nonsusceptibility among PCV7 serotypes (range, 65%-75%) and non-PCV7 serogroups (range, 11%-27%) did not significantly change overall. Although no change was detected among isolates collected from children with spontaneous drainage, the percentage of pneumococci recovered at the time of myringotomy and/or tympanostomy tube placement that were nonresistant to penicillin decreased over time (from 73% in 1999 to 53% in 2002; P = .03). All of the serotype 3 strains were genetically related, whereas 88% of the isolates that were either serotype 19F or serotype 23F were related to 1 of 3 international clones. CONCLUSIONS Among children with AOM, the proportion of cases due to non-PCV7 serogroups increased, vaccine-related serotypes increased, and serotype 19F remained unchanged. Although a decrease in the proportion of cases due to PNSP occurred among children who required myringotomy and/or tympanostomy tube placement, the proportion of PNSP remained unchanged overall and among children with spontaneous drainage. Because future trends in the susceptibility patterns of pneumococcal isolates recovered from children with AOM are not easy to predict, continued surveillance is essential.

Epidemiology of Acute Otitis Media Caused by Streptococcus pneumoniae Before and After Licensure of the 7-Valent Pneumococcal Protein Conjugate Vaccine

We studied, by pulsed-field gel electrophoresis, multilocus sequence typing and penicillin-binding protein 2b amplicon-restriction profiles, pneumococcal isolates recovered from children with acute otitis media during 1 January-31 December 1999 and 2001. The proportion of nonvaccine serogroups increased from 14.8% (13/88) to 36.5% (23/63) from 1999 to 2001 (P<.01). Among children who received at least 2 doses of the pneumococcal 7-valent protein conjugate (PNC7) vaccine, 46.7% (7/15) of the isolates had nonvaccine serogroups, compared with 20.8% (26/125) of the isolates from children who did not receive the PNC7 vaccine (P=.05). Overall, the serogroups involved in capsular switching were 6-19-NT, 6-14-35, 15-19, and the 19-Spanish 23F clone. In 1999 and 2001, 30.8% (4/13) and 26.1% (6/23) of the nonvaccine serogroups were implicated in capsular switching, respectively. Continued surveillance will be of great importance as the distribution of the PNC7 vaccine increases.

Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil

ABSTRACT Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a β-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to β-lactams, aminoglycosides, and fluoroquinolones. The β-lactam resistance was conferred by the production of the metallo-β-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed with the semisynthetic aminoglycoside arbekacin, raising suspicion of 16S rRNA methylase production. A cloning experiment subsequently revealed the presence of a novel 16S rRNA methylase, RmtD, which accounted for the high-level resistance to all 4,6-disubstituted deoxystreptamine aminoglycosides, such as amikacin, tobramycin, and gentamicin. RmtD shared a moderate degree of identity with RmtA, another 16S rRNA methylase that was initially reported to occur in P. aeruginosa in Japan in 2003. This is the first identification of aminoglycoside resistance mediated by a 16S rRNA methylase in South America. This is also the first report to document coproduction of a metallo-β-lactamase and a 16S rRNA methylase, a combination that would severely compromise therapeutic options for the infected patients.

Epidemiological Profile of Linezolid-Resistant Coagulase-Negative Staphylococci

BACKGROUND Surveillance studies have shown that <0.1% of coagulase-negative staphylococci are linezolid resistant; however, at our institution, 4% of such organisms were found to be resistant. We investigated the risk factors for and the epidemiological profile of linezolid-resistant coagulase-negative staphylococci. METHODS Susceptibility testing and pulsed-field gel electrophoresis were performed to analyze the genetic relatedness of both linezolid-resistant and linezolid-susceptible isolates. Clinical data were retrieved from medical records, and a case-case-control study was performed to identify unique risk factors for linezolid resistance. RESULTS Isolates recovered from 25 patients with linezolid-resistant coagulase-negative staphylococci were examined; all but 1 of the isolates were identified as Staphylococcus epidermidis, and all but 1 had a minimum inhibitory concentration of linezolid of >256 microg/mL. Pulsed-field gel electrophoresis showed that 21 (84%) of 25 linezolid-resistant isolates exhibited genetic relatedness, whereas linezolid-susceptible isolates were of diverse clones. Unique, independent predictors of linezolid resistance included receipt of linezolid in the 3 months preceding isolation of the coagulase-negative staphylococci (odds ratio, 20.6; 95% confidence interval, 5.8-73.0). CONCLUSION Linezolid-resistant coagulase-negative staphylococci have emerged at our institution and are predominately of a single clone. We believe that the most likely scenario to explain this emergence is that person-to-person spread of linezolid-resistant coagulase-negative staphylococci led to establishment of skin colonization with the strain. Subsequent use of linezolid was followed by selection of the linezolid-resistant strain, which then became the dominant skin flora. The potential for a parallel scenario involving clonal dissemination followed by selection of linezolid-resistant methicillin-resistant Staphylococcus aureus is a real possibility.

High Prevalence of Metallo-β-Lactamase and 16S rRNA Methylase Coproduction among Imipenem-Resistant Pseudomonas aeruginosa Isolates in Brazil

ABSTRACT Rates of metallo-β-lactamase and 16S rRNA methylase production were investigated in 51 imipenem-resistant Pseudomonas aeruginosa clinical isolates collected from hospitals in São Paulo, Brazil. Of them, 57% and 75% produced SPM-1 and RmtD, respectively. Of note, 51% produced both enzymes, suggesting that their coproduction is already common in this geographic area.

Identification of 16S rRNA Methylase-Producing Acinetobacter baumannii Clinical Strains in North America

ABSTRACT Five highly amikacin-resistant Acinetobacter baumannii isolates were collected at a medical center in Pennsylvania. The aminoglycoside resistance was due to the production of the 16S rRNA methylase ArmA. Two of the isolates coproduced OXA-23 β-lactamase and were highly resistant to carbapenems as well. The isolates were genetically closely related by pulsed-field gel electrophoresis.

Web-based Investigation of Multistate Salmonellosis Outbreak

We investigated a large outbreak of Salmonella enterica serotype Javiana among attendees of the 2002 U.S. Transplant Games, including 1,500 organ transplant recipients. Web-based survey methods identified pre-diced tomatoes as the source of this outbreak, which highlights the utility of such investigative tools to cope with the changing epidemiology of foodborne diseases.

KPC type beta-lactamase, rural Pennsylvania

To the Editor: Rural counties have been defined as those lacking a metropolitan center that has a population >50,000 persons (1). Little is known about antimicrobial drug resistance in such communities in the United States. Stevenson and colleagues (2) recently evaluated antimicrobial drug–resistant gram-positive infections in rural hospitals in Idaho and Utah. These researchers found that both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci occurred in such settings, although some of the MRSA strains were probably community associated. Comparable studies on multidrug-resistant gram-negative infections have not been performed, to our knowledge. Klebsiella pneumoniae producing a broad-spectrum β-lactamase, KPC, has been described in tertiary care centers and other metropolitan hospitals in New York City. Examples have also been found in similar settings in Boston, New Jersey, Maryland, and North Carolina (3–5). The carbapenems (such as imipenem and meropenem) are typically the most active antimicrobial agents against the Enterobacteriaceae. The KPC β-lactamases inactivate carbapenems and all other β-lactam antimicrobial drugs. Unfortunately, bacteria producing the KPC type β-lactamases are typically also resistant to trimethoprim/sulfamethoxazole, quinolones, and aminoglycosides, thereby making these pathogens truly multidrug resistant. We describe a patient with KPC-producing K. pneumoniae in a rural setting in central-west Pennsylvania. The case highlights the potential for multidrug-resistant gram-negative organisms to occur outside their previously recognized settings in large metropolitan centers. The patient was a 76-year-old woman who lived alone, closely attended by her daughter, in a small, central Pennsylvania community, 95 miles from a metropolitan center with a population of >50,000. Her medical history included a seizure disorder, hypertension, osteoarthritis of the knees, obesity, osteoporosis, and total hysterectomy. A month before isolation of the KPC-producing K. pneumoniae, the patient had a 3-day hospital admission to a 200-bed hospital in the nearest metropolitan center (population 7,000) after a fall. She was discharged to a local nursing home for rehabilitation. She is not known to have visited or been hospitalized in New York, Philadelphia, or New Jersey, nor did she share a room with a patient known to have been hospitalized in these areas. She had no known animal contact. She had received trimethoprim/sulfamethoxazole and levofloxacin for treatment for urinary tract infections in the month before the KPC-producing strain was isolated. She was readmitted to the 200-bed hospital with pyelonephritis in August 2005. Cultures of urine grew K. pneumoniae; the organism was resistant to all β-lactam antimicrobial drugs tested, including cefepime, ceftriaxone, piperacillin/tazobactam, imipenem, fluoroquinolones, trimethoprim/sulfamethoxazole, gentamicin, and tobramycin. The patient received therapy with amikacin in combination with cefepime, ertapenem, or tigecycline at different times over the following 4 weeks. Her symptoms improved, although her urine remained colonized with the multidrug-resistant K. pneumoniae. In October 2005, Clostridium difficile infection developed, accompanied by deep venous thrombosis and gastrointestinal bleeding, and the patient died. Multiple blood cultures collected before her death were negative, although the urine was persistently colonized with the multidrug-resistant K. pneumoniae. The organism was referred to a research laboratory in a metropolitan center ≈100 miles away. Molecular analysis of the mechanisms of resistance was performed by using previously described methods (6). This analysis showed that the K. pneumoniae isolate produced the extended-spectrum β-lactamase (ESBL) SHV-11 and the carbapenemase KPC-2. Since community-associated ESBL-producing organisms have been described in Canada and Europe (7,8), acquisition or in vivo development of ESBL and KPC-producing strains could have occurred outside of the healthcare setting. More likely, the patient acquired her almost completely resistant gram-negative organism in the rural hospital or her local nursing home. To our knowledge, no other clinical isolates with the same antimicrobial phenotype have been seen in patients in either setting before or after the patients admission. An unsuspected reservoir of patients colonized with antimicrobial drug–resistant gram-negative organisms may exist (9). Ideally, an epidemiologic investigation at both the hospital and nursing home would have been performed, but facilities for an investigation involving use of selective microbiologic media and assessment of gastrointestinal carriage of resistant organisms are not typically available in a rural setting. Indeed, most rural hospitals do not even use routine diagnostic tests for detecting resistant gram-negative organisms such as ESBL producers (10). Although much attention has been focused on the progression of antimicrobial drug resistance in gram-positive organisms, the development of alternative antimicrobial agents such as linezolid and daptomycin may mitigate the disastrous scenario of complete resistance to all commercially available antimicrobial agents. However, few drugs are active against multidrug-resistant gram-negative pathogens, and enhanced measures are needed to prevent spread of these organisms. A greater understanding of the modes of spread and acquisition of these organisms is essential for effective control of this problem. We have reported just 1 case of infection with an almost completely resistant gram-negative organism. This case expands the known geographic spread of organisms with this resistance problem. This case also underscores the importance of studying the epidemiology of antimicrobial drug resistance in gram-negative organisms in the rural setting as well as in large metropolitan centers. Dissemination of knowledge regarding appropriate antimicrobial drug susceptibility testing for resistant organisms is also needed.