Community-associated extended-spectrum ß-lactamase-producing Escherichia coli infection in the United States.

Background. The occurrence of community-associated infections due to extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli has been recognized as a major clinical problem in Europe and other regions. Methods. We conducted a prospective observational study to examine the occurrence of community-associated infections due to ESBL-producing E. coli at centers in the United States. Five academic and community hospitals and their affiliated clinics participated in this study in 2009 and 2010. Sites of acquisition of the organisms (community-associated or healthcare-associated), risk factors, and clinical outcome were investigated. Screening for the global epidemic sequence type (ST) 131 and determination of the ESBL types was conducted by polymerase chain reaction and sequencing. Results. Of the 291 patients infected or colonized with ESBL-producing E. coli as outpatients or within 48 hours of hospitalization, 107 (36.8%) had community-associated infection (81.5% of which represented urinary tract infection), while the remainder had healthcare-associated infection. Independent risk factors for healthcare-associated infection over community-associated infection were the presence of cardiovascular disease, chronic renal failure, dementia, solid organ malignancy, and hospitalization within the previous 12 months. Of the community-associated infections, 54.2% were caused by the globally epidemic ST131 strain, and 91.3% of the isolates produced CTX-M-type ESBL. Conclusions. A substantial portion of community-onset, ESBL-producing E. coli infections now occur among patients without discernible healthcare-associated risk factors in the United States. This epidemiologic shift has implications for the empiric management of community-associated infection when involvement of E. coli is suspected.

The role of horizontal gene transfer in the dissemination of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates in an endemic setting.

The contribution of horizontal gene transmission (HGT) in the emergence and spread of extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria during periods of endemicity is unclear. Over a 12-month period, rectal colonization with SHV-5- and SHV-12-producing Escherichia coli and Klebsiella pneumoniae was quantified among a cohort of residents in a long-term care facility. Demographic and clinical data were collected on colonized residents. Transferability of SHV-encoding plasmids and pulsed-field gel electrophoresis were performed to quantify the contribution of HGT and cross-transmission, respectively. A total of 25 (12%) of 214 enrolled patients were colonized with 11 SHV-5- and 17 SVH-12-producing E. coli and K. pneumoniae. Clonally related isolates were detected among multiple residents residing on the same and different wards. Among 12 clonally distinct isolates, HGT of SHV-5- and SHV-12-encoding plasmids was identified among 6 (50%) isolates. HGT among clonally distinct strains contributes to the transmission dynamics of these ESBL-producing Gram-negative bacteria and should be considered when evaluating the spread of these pathogens.

Features of infections due to Klebsiella pneumoniae carbapenemase-producing Escherichia coli: emergence of sequence type 131.

BACKGROUND: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become endemic in many US hospitals. On the other hand, KPC-producing Escherichia coli remains rare. METHODS: We studied infection or colonization due to KPC-producing E. coli identified at our hospital between September 2008 and February 2011. A case-control study was conducted to document clinical features associated with this organism. Susceptibility testing, sequencing of ß-lactamase genes, pulsed-field gel electrophoresis, multilocus sequence typing, and plasmid analysis were performed for characterization of the isolates. RESULTS: Thirteen patients with KPC-producing E. coli were identified. The patients had multiple comorbid conditions and were in hospital for variable periods of time before KPC-producing E. coli was identified. The presence of liver diseases was independently associated with recovery of KPC-producing E. coli when compared with extended-spectrum ß-lactamase-producing E. coli. The isolates showed variable susceptibility to carbapenems. Seven isolates belonged to sequence type (ST) 131, which is the international epidemic, multidrug-resistant clone, but their plasmid profiles were diverse. KPC-producing organisms other than E. coli were isolated within 1 month from 5 of the patients. The KPC-encoding plasmids were highly related in 3 of them, suggesting the occurrence of their interspecies transfer. CONCLUSIONS: KPC-producing E. coli infections occur in severely ill patients who are admitted to the hospital. Acquisition of the KPC-encoding plasmids by the ST 131 clone, reported here for the first time to our knowledge in the United States, seems to represent multiple independent events. These plasmids are often shared between E. coli and other species.

Clinical and microbiologic characteristics of cephalosporin-resistant Escherichia coli at three centers in the United States.

We investigated the clinical and microbiologic features of 300 cases of cephalosporin-resistant Escherichia coli producing extended-spectrum ß-lactamase (ESBL) or plasmid-mediated AmpC ß-lactamase (pAmpC) at three medical centers in the United States. Solid-organ malignancy, connective tissue disease, and a recent history of surgery were more common among pAmpC-producing cases (n = 49), whereas urinary catheter at enrollment, diabetes, and hospitalization in the past year were more common among ESBL-producing cases (n = 233). The factors independently associated with clinical outcome were the following: the presence of cardiovascular disease (odds ratio [OR], 2.88; 95% confidence interval [CI], 1.29 to 6.43), intra-abdominal infection (OR, 6.35; 95% CI, 1.51 to 26.7), other or multiples sources of infection (OR, 8.12; 95% CI, 2.3 to 28.6), age of 65 years or greater (OR, 0.43; 95% CI, 0.2 to 0.95), favorable baseline health status (OR, 0.39; 95% CI, 0.16 to 0.95), and appropriate empirical antimicrobial therapy given in the first 72 h (OR, 0.42; 95% CI, 0.20 to 0.88). ß-Lactamase genes responsible for cephalosporin resistance were identified in 291 cases. CTX-M-type ESBLs accounted for 72.0%. Of those, 88.0% were CTX-M-15. The next most common type was CMY-type pAmpC (16.7%), followed by SHV- and TEM-type ESBLs (6.3 and 1.3%, respectively). Seven cases (2.3%) had KPC-type ß-lactamase. Ertapenem, imipenem, meropenem, doripenem, piperacillin-tazobactam, amikacin, nitrofurantoin, and tigecycline were highly active, with greater than 90% of the isolates being susceptible. Cefepime was less active, with only 74.2% being susceptible due to the predominance of CTX-M-15. These findings have implications in the selection of appropriate empirical therapy when infection due to cephalosporin-resistant E. coli is suspected.

Treatment outcome of bacteremia due to KPC-producing Klebsiella pneumoniae: superiority of combination antimicrobial regimens.

Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been associated with serious infections and high mortality. The optimal antimicrobial therapy for infection due to KPC-producing K. pneumoniae is not well established. We conducted a retrospective cohort study to evaluate the clinical outcome of patients with bacteremia caused by KPC-producing K. pneumoniae. A total of 41 unique patients with blood cultures growing KPC-producing K. pneumoniae were identified at two medical centers in the United States. Most of the infections were hospital acquired (32; 78%), while the rest of the cases were health care associated (9; 22%). The overall 28-day crude mortality rate was 39.0% (16/41). In the multivariate analysis, definitive therapy with a combination regimen was independently associated with survival (odds ratio, 0.07 [95% confidence interval, 0.009 to 0.71], P = 0.02). The 28-day mortality was 13.3% in the combination therapy group compared with 57.8% in the monotherapy group (P = 0.01). The most commonly used combinations were colistin-polymyxin B or tigecycline combined with a carbapenem. The mortality in this group was 12.5% (1/8). Despite in vitro susceptibility, patients who received monotherapy with colistin-polymyxin B or tigecycline had a higher mortality of 66.7% (8/12). The use of combination therapy for definitive therapy appears to be associated with improved survival in bacteremia due to KPC-producing K. pneumoniae.

Molecular epidemiology of carbapenem-nonsusceptible Acinetobacter baumannii in the United States.

Acinetobacter baumannii is emerging as an important nosocomial pathogen worldwide. We report molecular epidemiology of 65 carbapenem-nonsusceptible A. baumannii isolates identified from hospitals in New York, Pennsylvania, Florida, Missouri, Nevada, and California between 2008 and 2009. All isolates were subjected to pulsed-field gel electrophoresis (PFGE). Select isolates then underwent multilocus sequence typing (MLST). While the PFGE patterns tended to cluster within each hospital, sequence types (STs) belonging to the clonal complex 92 (CC92) and the pan-European clonal lineage II (EUII; worldwide clonal lineage 2) were predominant in all hospitals. Of them, ST122 and ST208 were the most common and were found in four of the six hospitals. Isolates belonging to the pan-European clonal lineages I and III were identified in one hospital each. Carbapenemase-encoding genes bla(OXA-23) and/or ISAba1-bla(OXA-51-like) were present among the majority of isolates. These findings suggest that carbapenem-nonsusceptible A. baumannii isolates found in U.S. hospitals constitute part of the global epidemic driven by CC92, but have unique STs other than ST92, which may be spreading by means of patient transfer between health care facilities within the United States.

Colistin-resistant, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae belonging to the international epidemic clone ST258.

Five cases of infection due to colistin-resistant, Klebsiella pneumoniae carbapenemase-producing K. pneumoniae belonging to the international epidemic clone ST258 occurred over a 4-month period. These cases likely represented both emergence of resistance and transmission of resistant organism. The colistin-resistant isolates were able to persist in the absence of selective pressure in vitro.

Extended-spectrum AmpC cephalosporinase in Acinetobacter baumannii: ADC-56 confers resistance to cefepime.

ADC-56, a novel extended-spectrum AmpC (ESAC) ß-lactamase, was identified in an Acinetobacter baumannii clinical isolate. ADC-56 possessed an R148Q change compared with its putative progenitor, ADC-30, which enabled it to hydrolyze cefepime. Molecular modeling suggested that R148 interacted with Q267, E272, and I291 through a hydrogen bond network which constrained the H-10 helix. This permitted cefepime to undergo conformational changes in the active site, with the carboxyl interacting with R340, likely allowing for better binding and turnover.

PME-1, an extended-spectrum ß-lactamase identified in Pseudomonas aeruginosa.

A novel extended-spectrum ß-lactamase (ESBL) was identified in a Pseudomonas aeruginosa clinical isolate obtained from a patient admitted to a hospital in Pennsylvania in 2008. The patient had a prolonged hospitalization in a hospital in Dubai, United Arab Emirates, before being transferred to the United States. The novel ESBL, designated PME-1 (Pseudomonas aeruginosa ESBL 1), is a molecular class A, Bush-Jacoby-Medeiros group 2be enzyme and shared 50, 43, and 41% amino acid identity with the L2 ß-lactamase of Stenotrophomonas maltophilia, CTX-M-9, and KPC-2, respectively. PME-1 conferred clinically relevant resistance to ceftazidime, cefotaxime, cefepime, and aztreonam in P. aeruginosa PAO1 but not to carbapenems. Purified PME-1 showed good hydrolytic activity against ceftazidime, cefotaxime, and aztreonam, while activity against carbapenems and cefepime could not be measured. PME-1 was inhibited well by ß-lactamase inhibitors, including clavulanic acid, sulbactam, and tazobactam. The bla(PME-1) gene was carried by an approximately 9-kb plasmid and flanked by tandem ISCR24 elements.

High mortality rates among solid organ transplant recipients infected with extensively drug-resistant Acinetobacter baumannii: using in vitro antibiotic combination testing to identify the combination of a carbapenem and colistin as an effective treatment regimen.

Extensively drug-resistant (XDR) Acinetobacter baumannii infections caused 91% (10/11) mortality in transplant recipients. Isolates were colistin-susceptible initially, but susceptibility decreased during therapy in 40% (4/10). We tested antibiotic combinations against XDR Acinetobacter in vitro and demonstrated positive interactions for carbapenem-colistin. Subsequently, 80% (4/5) of transplant patients were treated successfully with carbepenem-colistin regimens.

Screening for Acinetobacter baumannii colonization by use of sponges.

There is currently no consensus method for the active screening of Acinetobacter baumannii. The use of swabs to culture nostrils, pharynx, and skin surface of various anatomical sites is known to yield less-than-optimal sensitivity. In the present study, we sought to determine whether the use of sterile sponges to sample large areas of the skin would improve the sensitivity of the detection of A. baumannii colonization. Forty-six patients known to be colonized with A. baumannii, defined by a positive clinical culture for this organism as defined by resistance to more than two classes of antimicrobials, participated in the study. The screening sites included the forehead, nostrils, buccal mucosa, axilla, antecubital fossa, groin, and toe webs with separate rayon swabs and the forehead, upper arm, and thigh with separate sponges. Modified Leeds Acinetobacter medium (mLAM) agar plates that contained vancomycin and either aztreonam or ceftazidime were used as the selective medium. An enrichment culture grown overnight substantially increased the sensitivity for most sites. The sensitivity ranged between 69.6 and 82.6% for individual sponge sites and 21.7 to 52.2% for individual swab sites when mLAM plates with ceftazidime were inoculated after a 24-h enrichment period. The sponge and swab sites with the best sensitivity were the leg and the buccal mucosa, respectively (82.6% and 52.2%; P = 0.003). The combined sensitivity for the upper arm and leg with a sponge was 89.1%. The novel screening method using sterile sponges was easy to perform and achieved excellent sensitivity for the detection of A. baumannii colonization.

Clinical characteristics of bloodstream infections due to ampicillin-sulbactam-resistant, non-extended- spectrum-beta-lactamase-producing Escherichia coli and the role of TEM-1 hyperproduction.

Ampicillin-sulbactam is commonly used as an empirical therapy for invasive infections where Escherichia coli is a potential pathogen. We evaluated the clinical and microbiologic characteristics of bloodstream infection due to E. coli, with focus on cases that were nonsusceptible to ampicillin-sulbactam and not producing extended-spectrum ß-lactamase (ESBL). Of a total of 357 unique bacteremic cases identified between 2005 and 2008, 111 (31.1%) were intermediate or resistant to ampicillin-sulbactam by disk testing. In multivariate analysis, a history of liver disease, organ transplant, peptic ulcer disease, and prior use of ampicillin-sulbactam were independent risk factors for bloodstream infection with ampicillin-sulbactam-nonsusceptible E. coli. Among cases that received ampicillin-sulbactam as an empirical therapy, an early clinical response was observed in 65% (22/34) of susceptible cases but in only 20% (1/5) of nonsusceptible cases. Among 50 ampicillin-sulbactam-resistant isolates examined, there was no clonal relatedness and no evidence of production of inhibitor-resistant TEM (IRT). Instead, the resistance was attributed to hyperproduction of TEM-1 ß-lactamase in the majority of isolates. However, promoter sequences of bla(TEM-1) did not predict resistance to ampicillin-sulbactam. While the plasmid copy number did not differ between representative resistant and susceptible isolates, the relative expression of bla(TEM-1) was significantly higher in two of three resistant isolates than in three susceptible isolates. These results suggest high-level bla(TEM-1) expression as the predominant cause of ampicillin-sulbactam resistance and also the presence of yet-unidentified factors promoting overexpression of bla(TEM-1) in these isolates.

Risk factors and outcome of extended-spectrum ß-lactamase-producing Enterobacter cloacae bloodstream infections.

Enterobacter cloacae is a major nosocomial pathogen that causes serious infections, including bloodstream infections (BSIs). The clinical significance of extended-spectrum ß-lactamase (ESBL) production in E. cloacae is not well established. A multicentre, retrospective, cohort study was conducted to identify clinical characteristics of patients with E. cloacae BSI. ESBL production was confirmed by genotypic methods. A total of 159 patients with E. cloacae BSI were identified at three medical centres in north-eastern USA. Amongst them, 16 patients (10.1%) harboured ESBL-producing E. cloacae. Independent risk factors for ESBL production included admission from a nursing home, the presence of a gastrostomy tube and history of transplant. For the outcome analysis, 15 consecutive patients who had ESBL-producing E. cloacae BSI prior to the study were included. Amongst the 31 patients with ESBL-producing E. cloacae, 8, 9, 4 and 2 patients received a carbapenem, cefepime, piperacillin/tazobactam and ciprofloxacin, respectively, as initial therapy. All patients who received a carbapenem (n=8) were alive at 28 days, whereas 7 (38.9%) of 18 patients who received a non-carbapenem antibiotic did not survive (P=0.06). Clinical failure at 96 h was observed in 2 (25.0%) of 8 patients who received a carbapenem and in 14 (77.8%) of 18 patients who received a non-carbapenem antibiotic (P=0.03). Pulsed-field gel electrophoresis showed little clonality amongst the study isolates. The majority of isolates produced SHV-type ESBL, whereas two isolates produced CTX-M-type ESBL. Initial therapy with a carbapenem appears to be associated with improved clinical outcome in BSI due to ESBL-producing E. cloacae.

Identification of diverse OXA-40 group carbapenemases, including a novel variant, OXA-160, from Acinetobacter baumannii in Pennsylvania.

Three Acinetobacter baumannii isolates that possess OXA-40 group carbapenemase genes were identified. They belonged to novel sequence types (ST122, ST123, and ST124) and harbored bla(OXA-160), bla(OXA-72), and bla(OXA-40), respectively. OXA-160 is a novel variant of OXA-40 with a P227S substitution. An isogenic Escherichia coli clone producing OXA-160 was more susceptible to carbapenems than a clone producing OXA-40. The genetic environment of bla(OXA-160) and bla(OXA-40) beyond the putative XerC/XerD recombination sites was distinct from the scaffold reported previously.

Enhancing resistance to cephalosporins in class C beta-lactamases: impact of Gly214Glu in CMY-2.

The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Omega loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C beta-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (k(cat)/K(m)) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k(cat)/K(m) against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone beta-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both beta-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is beta-lactamase-catalyzed elimination of the C(6) hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new beta-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO(3)(2-). Molecular modeling and dynamics simulations suggest that the Omega loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis. When it is docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 beta-lactamases.

Interspecies spread of Klebsiella pneumoniae carbapenemase gene in a single patient.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, Escherichia coli, and Serratia marcescens were sequentially identified in a patient who underwent small bowel transplantation. Molecular typing and plasmid analysis suggested that the KPC gene was acquired by E. coli, most likely from K. pneumoniae, and was subsequently transferred to S. marcescens.