Extended-Spectrum-β-Lactamase-Producing Escherichia coli as Intestinal Colonizers in the German Community

We determined the presence of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli among 3,344 study participants from the German community. Intestinal colonization was detected in 211 persons (6.3%), without significant differences among the different age groups. The majority (95.2%) of isolates harbored CTX-M-type ESBL, with CTX-M-15 (46%) and CTX-M-1 (24.2%) as the most common types. The finding of ESBL producers and one isolate additionally producing carbapenemase OXA-244 indicates a risk of dissemination of resistant bacteria outside the hospitals.     

Multicenter Retrospective Study of Cefmetazole and Flomoxef for Treatment of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Bacteremia

The efficacy of cefmetazole and flomoxef (CF) for the treatment of patients with extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) bacteremia (ESBL-CF group) was compared with that of carbapenem treatment for ESBL-EC patients (ESBL-carbapenem group) and with that of CF treatment in patients with non-ESBL-EC bacteremia (non-ESBL-CF group). Adult patients treated for E. coli bacteremia in four hospitals were retrospectively evaluated. The 30-day mortality rates in patients belonging to the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were compared as 2 (empirical and definitive therapy) cohorts. The adjusted hazard ratios (aHRs) for mortality were calculated using Cox regression models with weighting according to the inverse probability of propensity scores for receiving CF or carbapenem treatment. The empirical-therapy cohort included 104 patients (ESBL-CF, 26; ESBL-carbapenem, 45; non-ESBL-CF, 33), and the definitive-therapy cohort included 133 patients (ESBL-CF, 59; ESBL-carbapenem, 54; non-ESBL-CF, 20). The crude 30-day mortality rates for patients in the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were, respectively, 7.7%, 8.9%, and 3.0% in the empirical-therapy cohort and 5.1%, 9.3%, and 5.0% in the definitve-therapy cohort. In patients without hematological malignancy and neutropenia, CF treatment for ESBL-EC patients was not associated with mortality compared with carbapenem treatment (empirical-therapy cohort: aHR, 0.87; 95% confidence interval [CI], 0.11 to 6.52; definitive therapy cohort: aHR, 1.04; CI, 0.24 to 4.49). CF therapy may represent an effective alternative to carbapenem treatment for patients with ESBL-EC bacteremia for empirical and definitive therapy in adult patients who do not have hematological malignancy and neutropenia.Bacteremia caused by Escherichia coli is a common and significant problem in both community and health care settings (1). In recent years, the prevalence of extended-spectrum β-lactamase-producing E. coli (ESBL-EC) has increased dramatically worldwide. Effective treatment of ESBL-EC bacteremia has become challenging because ESBL-EC is usually resistant to cephalosporins, the first-line drug used for E. coli infections (2). At least partially due to the delay in effective treatment regimens, bacteremia caused by ESBL-EC is associated with a higher mortality rate than that caused by non-ESBL-EC (3). Currently, the standard therapy for ESBL-EC bacteremia is virtually limited to carbapenems (4, 5). Alternate therapies involving fluoroquinolones, sulfamethoxazole-trimethoprim, and aminoglycosides offer limited efficacy due to frequent coresistance mechanisms in ESBL-EC (2). To prevent carbapenem overuse and spread of carbapenem-resistant Enterobacteriaceae, alternative drug regimens are needed (6).β-Lactam/β-lactamase inhibitors and cephamycins (i.e., cefmetazole, cefoxitin, cefotetan, moxalactam, and flomoxef) are usually stable to hydrolysis by ESBLs (2). The MICs of β-lactam/β-lactamase inhibitors can increase as the inoculum increases (7); however, cephamycins (cefotetan and flomoxef) exert in vitro antibacterial activity even in the presence of high inocula (8, 9). Clinical data for β-lactam/β-lactamase inhibitors obtained with a large cohort of patients by using propensity score analysis have been published, but the conclusions are inconsistent (10, 11). In Japan, cefmetazole and flomoxef (CF) are frequently used to treat intra-abdominal infections or as prophylaxis for surgery. Thus, Japanese clinicians have sometimes used CF therapy for sepsis or bacteremia as empirical therapy or as definitive therapy when susceptibility was confirmed. However, clinical data evaluating the effectiveness of cephamycins for ESBL-EC bacteremia have not been reported yet.In this multicenter retrospective study using propensity score analysis, we evaluated the effectiveness of empirical and definitive CF treatments for ESBL-EC bacteremia in comparison with standard carbapenem therapy. We also evaluated patients with non-ESBL-EC bacteremia who were treated with a CF regimen to investigate potential associations between ESBL production and patient outcomes when E. coli bacteremia was treated with CF therapy.     

Effect of Ceftriaxone on the Outcome of Murine Pyelonephritis Caused by Extended-Spectrum-β-Lactamase-Producing Escherichia coli

Urinary tract infections (UTIs) due to extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae in children are becoming more frequent, and they are commonly treated initially with a second- or third-generation cephalosporin. We developed a murine model of ascending UTI caused by ESBL-producing Escherichia coli. Using this model, we investigated the renal bacterial burden, interleukin-6 (IL-6) expression, and histopathological alterations caused by ESBL- and non-ESBL-producing bacteria after 1, 2, or 6 days with or without ceftriaxone therapy. The renal bacterial burden, IL-6 concentration, and histological inflammatory lesions were not significantly different between mice infected with ESBL- and non-ESBL-producing bacteria without treatment at any of the time points examined. Following ceftriaxone administration, the bacterial burden was eliminated in the kidneys of mice infected with ESBL- and non-ESBL-producing bacteria on the 6th postinfection day. The histological analysis demonstrated that among mice treated with ceftriaxone, those infected with ESBL-producing bacteria had more profound renal alterations than those infected with non-ESBL-producing bacteria on the 6th day (P < 0.001). In comparison, microbiological outcomes did not differ significantly between mice infected with ESBL- and non-ESBL-producing bacteria at any of the time points examined. The effectiveness of ceftriaxone in mice with UTIs due to ESBL-producing E. coli may have therapeutic implications; it is, however, hampered by limited activity on the histopathological lesions, a finding that needs further investigation.     

Molecular Features of Community-Associated Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains in the United States

We characterized 30 community-associated extended-spectrum-β-lactamase-producing Escherichia coli isolates collected from five hospitals in the United States. Nineteen sequence types were identified. All sequence type 131 (ST131) isolates had the fimH30 allele. IncFII-FIA-FIB was the most common replicon type among the bla_CTX-M-carrying plasmids, followed by IncFII-FIA and IncA/C. Restriction analysis of the IncFII-FIA-FIB and IncFII-FIA plasmids yielded related profiles for plasmids originating from different hospitals. The plasmids containing bla_CTX-M or bla_SHV were stably maintained after serial passages.     

Oral Treatment Options for Ambulatory Patients with Urinary Tract Infections Caused by Extended-Spectrum-β-Lactamase-Producing Escherichia coli

An increase in extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli has been observed in outpatient settings. Consequently, 100 ESBL-positive E. coli isolates from ambulatory patients with clinically confirmed urinary tract infections were collected by a single laboratory between October 2004 and January 2008. Antimicrobial susceptibility testing was carried out using the oral antibiotics fosfomycin, pivmecillinam, and nitrofurantoin and the parenteral antibiotic ertapenem. Susceptibility rates indicate that fosfomycin (97%), nitrofurantoin (94%), and pivmecillinam (85%) could be considered important oral treatment options.Escherichia coli is the most common pathogen of bacterial infections worldwide. As many as 80% of urinary tract infections (UTIs) are caused by E. coli. In 1980, resistance to extended-spectrum cephalosporins was found for the first time in Enterobacteriaceae showing no chromosomally encoded AmpC overexpression. This newly detected plasmid-encoded resistance was selected by the frequent use of cephalosporins. These bacterial enzymes have been named extended-spectrum β-lactamases (ESBL) due to their capacity to inactivate practically all cephalosporins (21). ESBL-producing phenotypes of the family of Enterobacteriaceae were primarily considered multiresistant organisms originating in hospitals. In recent years, an increase of such ESBL producers has been observed in outpatient settings, especially related to UTIs, reducing the treatment options to a limited number of antibiotics (2, 3, 9, 14, 21). Of special concern are associated coresistances to other classes of antimicrobials, which aid the spreading of multiresistant isolates (12). CTX-M β-lactamase-producing Enterobacteriaceae, which are commonly found in outpatients and isolated from UTIs, are typically also resistant to quinolones, aminoglycosides, and sulfonamides, such as ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole, respectively (10, 16).The aim of our study was to evaluate antimicrobial agents which can be used in outpatient health care for the treatment of uncomplicated and complicated UTIs caused by ESBL-producing E. coli. For this purpose, we analyzed susceptibility rates of E. coli isolates from clinically significant UTIs to fosfomycin, pivmecillinam, nitrofurantoin, and ertapenem.Fosfomycin is a phosphor acid derivative produced by Streptomyces spp. It inhibits bacterial cell wall synthesis and impairs the adherence to urogenital mucosa. Stabilized with tromethamine, it can be orally administered as a single dose of 3 g for the treatment of UTIs (17). It is well tolerated, with negligible side effects, such as diarrhea and headache, and is applicable during pregnancy (6, 7).Pivmecillinam is a β-lactam antibiotic which works specifically on Enterobacteriaceae by binding to penicillin-binding protein 2 and inhibiting the bacterial cell wall synthesis. An orally administered, twice-a-day dose of 400 mg is recommended for the treatment of UTIs (8).Nitrofurantoin is a bactericidal drug. It is reduced by bacterial flavoproteins to reactive intermediates which inactivate or alter bacterial ribosomal proteins and other macromolecules. A 7-day, twice-daily administration of 100 mg is recommended (http://www.drugs.com/pro/nitrofurantoin-capsules.html).Ertapenem is a broad-spectrum β-lactam antibiotic that can be administered only parenterally, but it has a long half-life, allowing for a treatment dose of 1 g per day in outpatient health care (22).ESBL-producing E. coli isolates (n = 100) were collected consecutively (October 2004 to January 2008) from clinically certified UTIs. Ninety-eight specimens were submitted by attending general practitioners, while only two were derived from hospitals. Limited information was available concerning patients'' previous treatment with antibiotics, previous hospitalization, or risk factors for urinary tract infections. Patients'' ages ranged from 2 to 97 years (average mean age, 57.6 years). Seventy-eight percent of isolates derived from female patients, and 22% derived from male patients. Seven isolates came from patients in long-term-care facilities.E. coli isolates (n = 6,066) from UTIs with resistance to one or more antimicrobial agents were screened (Table ​(Table1).1). Resistance testing was carried out with the Vitek 2 system and the AST-N020 card (bioMerieux, Marcy l''Etoile, France) according to the manufacturer''s instructions. Isolates were considered presumptive ESBL producers when they exhibited MICs of cefotaxime, cefpodoxime, and ceftazidime that were ≥2 μg/ml. ESBL expression was confirmed by means of the double-disk synergy test with commercially available discs (Oxoid, Basingstoke, United Kingdom) according to 2007 CLSI guidelines (4). All verified ESBL-producing strains (n = 100) were tested for pivmecillinam, fosfomycin, and ertapenem. Susceptibility testing for all three substances was carried out with the agar diffusion test and the Etest. All results were evaluated according to the 2007 CLSI guidelines (4).

TABLE 1.

Proportions of ESBL-producing E. coli isolates susceptible to the antimicrobial agents examined

Impact of the MIC of Piperacillin-Tazobactam on the Outcome of Patients with Bacteremia Due to Extended-Spectrum-β-Lactamase-Producing Escherichia coli

We investigated the impact of the piperacillin-tazobactam MIC in the outcome of 39 bloodstream infections due to extended-spectrum-β-lactamase-producing Escherichia coli. All 11 patients with urinary tract infections survived, irrespective of the MIC. For other sources, 30-day mortality was lower for isolates with a MIC of ≤2 mg/liter than for isolates with a higher MIC (0% versus 41.1%; P = 0.02).     

Molecular Characteristics of Travel-Related Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from the Calgary Health Region

Extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli has recently emerged as a major risk factor for community-acquired, travel-related infections in the Calgary Health Region. Molecular characterization was done on isolates associated with infections in returning travelers using isoelectric focusing, PCR, and sequencing for bla_CTX-Ms, bla_TEMs, bla_SHVs, bla_OXAs, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequence typing (MLST). A total of 105 residents were identified; 6/105 (6%) presented with hospital-acquired infections, 9/105 (9%) with health care-associated community-onset infections, and 90/105 (86%) with community-acquired infections. Seventy-seven of 105 (73%) of the ESBL-producing E. coli isolates were positive for bla_CTX-M genes; 55 (58%) produced CTX-M-15, 13 (14%) CTX-M-14, six (6%) CTX-M-24, one (1%) CTX-M-2, one (1%) CTX-M-3, and one (1%) CTX-M-27, while 10 (10%) produced TEM-52, three (3%) TEM-26, 11 (11%) SHV-2, and four (4%) produced SHV-12. Thirty-one (30%) of the ESBL-producing E. coli isolates were positive for aac(6′)-Ib-cr, and one (1%) was positive for qnrS. The majority of the ESBL-producing isolates (n = 95 [90%]) were recovered from urine samples, and 83 (87%) were resistant to ciprofloxacin. The isolation of CTX-M-15 producers belonging to clone ST131 was associated with travel to the Indian subcontinent (India, Pakistan), Africa, the Middle East, and Europe, while clonally unrelated strains of CTX-M-14 and -24 were associated with travel to Asia. Our study suggested that clone ST131 coproducing CTX-M-15, OXA-1, TEM-1, and AAC(6′)-Ib-cr and clonally unrelated CTX-M-14 producers have emerged as important causes of community-acquired, travel-related infections.Escherichia coli producing CTX-M β-lactamases have become the most prevalent type of extended-spectrum β-lactamases (ESBLs) described during the last 5 years, especially from certain European and South American countries (4). Infections caused by bacteria producing these enzymes are not limited to the hospital setting, and their potential for spread beyond the hospital environment is an important public health concern (27). CTX-M-producing E. coli strains isolated from hospital and community sites often exhibited coresistance to trimethoprim-sulfamethoxazole, tetracycline, gentamicin, tobramycin, and ciprofloxacin (23). CTX-M enzymes have been associated with the presence of different qnr genes producing proteins named QnrA, QnrS, and QnrB that block the action of fluoroquinolone on bacterial topoisomerases as well as the production of a novel aminoglycoside-modifying enzyme, AAC(6′)-Ib, that has the additional ability to modify certain fluoroquinolones (23).Multidrug-resistant CTX-M-15-producing E. coli is emerging worldwide in a simultaneous fashion as an important pathogen causing community-onset and hospital-acquired infections (31). An identical clone named ST131 has been identified, using multilocus sequence typing (MLST), among CTX-M-15-producing E. coli isolates isolated from 2000 to 2006 from several countries, including Spain, France, Canada, Portugal, Switzerland, Lebanon, India, Kuwait, and South Korea (7, 22). This clone belongs to the highly virulent phylogenetic group B2 and harbors multidrug-resistant IncFII plasmids. This suggests that clone ST131 has emerged independently in different parts of the world due to ingestion of contaminated food/water sources and/or is being imported into various countries via returning travelers (22). Clone ST131 has also recently been described in the United Kingdom (19), Italy (3), Turkey (34), and the United States (16).A previous study from the Calgary Health Region has demonstrated that overseas travel is a significant risk factor for acquiring community-acquired infections due to ESBL-producing E. coli (20). This follow-up study was designed to characterize these isolates and to determine if fluoroquinolone resistance in these isolates is partly due to plasmid-mediated quinolone resistance determinants.(Results of this study were presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008.)     

Characterization of Fecal Extended-Spectrum-β-Lactamase-Producing Escherichia coli in a Remote Community during a Long Time Period

Carriage of extended-spectrum beta-lactamase-producing enterobacteria (ESBL-E) has increased in community settings. Little is known about their long-term evolution. French Guiana Amerindians living in a very remote village, already sampled in 2001 and 2006 for ESBL-E fecal carriage, were screened again in October 2010. Sociodemographic data and antibiotic intake data were collected during the previous year. ESBL-E strains collected in 2010 and their plasmid contents were typed. The results were compared to those of the previous campaigns. The prevalence of ESBL-E carriage in 2010 was 5.3%, whereas it was 8.0% and 3.2% in 2006 and 2001, respectively. As previously determined, no individual factor was associated with carriage, including personal antibiotic exposure. However, overall antibiotic use had decreased to a 0.67 treatments/subject/year in 2010 versus 1.09 in 2006 (P < 0.001), which supports the idea that population exposure to antibiotics impacts on ESBL-E community carriage rates. A wide diversity of ESBL Escherichia coli strains belonging to the A₀, A₁, B1, and D₂ phylogroups and producing the CTX-M-1, CTX-M-2, and CTX-M-8 enzymes were isolated. Despite the overall genetic diversity of the strains evaluated by repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing, two CTX-M-1-producing clones were found to have spread. In contrast, similar ESBL-bearing I1/Iγ plasmids were present in various strains both within and between carriers, suggesting high rates of plasmid transfer. Our results suggest that overall antibiotic exposure affects ESBL-E fecal carriage in the community. ESBL-E spread may be the result of both strain dissemination and the transfer of plasmids in intestinal microbiota.     

Acquisition of Extended-Spectrum β-Lactamases by Escherichia coli and Klebsiella pneumoniae in Gut Microbiota of Pilgrims during the Hajj Pilgrimage of 2013

We reported the acquisition of extended-spectrum-β-lactamase (ESBL)-producing bacteria in rectal samples of 129 pilgrims during the 2013 Hajj (pilgrimage to Makkah). When returning from the Hajj, there was a significant increase in the number of pilgrims carrying E. coli resistant to ceftriaxone (P = 0.008). The CTX-M gene was detected in rectal samples, with the detection rate increasing from 10.08% to 32.56% of samples after the Hajj (P < 0.001).     

Appropriateness of Empirical Treatment and Outcome in Bacteremia Caused by Extended-Spectrum-β-Lactamase-Producing Bacteria

We studied clinical characteristics, appropriateness of initial antibiotic treatment, and other factors associated with day 30 mortality in patients with bacteremia caused by extended-spectrum-β-lactamase (ESBL)-producing bacteria in eight Dutch hospitals. Retrospectively, information was collected from 232 consecutive patients with ESBL bacteremia (due to Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae) between 2008 and 2010. In this cohort (median age of 65 years; 24 patients were <18 years of age), many had comorbidities, such as malignancy (34%) or recurrent urinary tract infection (UTI) (15%). One hundred forty episodes (60%) were nosocomial, 54 (23%) were otherwise health care associated, and 38 (16%) were community acquired. The most frequent sources of infection were UTI (42%) and intra-abdominal infection (28%). Appropriate therapy within 24 h after bacteremia onset was prescribed to 37% of all patients and to 54% of known ESBL carriers. The day 30 mortality rate was 20%. In a multivariable analysis, a Charlson comorbidity index of ≥3, an age of ≥75 years, intensive care unit (ICU) stay at bacteremia onset, a non-UTI bacteremia source, and presentation with severe sepsis, but not inappropriate therapy within <24 h (adjusted odds ratio [OR], 1.53; 95% confidence interval [CI], 0.68 to 3.45), were associated with day 30 mortality. Further assessment of confounding and a stratified analysis for patients with UTI and non-UTI origins of infection did not reveal a statistically significant effect of inappropriate therapy on day 30 mortality, and these results were insensitive to the possible misclassification of patients who had received β-lactam–β-lactamase inhibitor combinations or ceftazidime as initial treatment. In conclusion, ESBL bacteremia occurs mostly in patients with comorbidities requiring frequent hospitalization, and 84% of episodes were health care associated. Factors other than inappropriate therapy within <24 h determined day 30 mortality.     

Pharmacological Study of Cefoxitin as an Alternative Antibiotic Therapy to Carbapenems in Treatment of Urinary Tract Infections Due to Extended-Spectrum-β-Lactamase-Producing Escherichia coli

Cefoxitin could be an alternative to carbapenems in extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL-EC) infections. However, pharmacological and clinical data regarding cefoxitin are limited. Using a recent pharmacological model and the MICs of ESBL-EC collected from pyelonephritis, we determined the probabilities to reach four pharmacological targets: free cefoxitin concentrations above the MIC during 50% and 100% of the administration interval (T>MIC = 50% and T>MIC = 100%, respectively) and free cefoxitin concentrations above 4× MIC during 50% and 100% of the administration interval (T>4MIC = 50% and T>4MIC = 100%, respectively). Cefoxitin could be used to treat ESBL-EC pyelonephritis, but administration modalities should be optimized according to MICs in order to reach pharmacological targets.     

Characterization and Sequence Analysis of Extended-Spectrum-β-Lactamase-Encoding Genes from Escherichia coli,Klebsiella pneumoniae,and Proteus mirabilis Isolates Collected during Tigecycline Phase 3 Clinical Trials

In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla_TEM, bla_SHV, bla_OXA, and bla_CTX genes from clinical strains. Isolates were also screened for AmpC genes of the bla_CMY, bla_ACT, bla_FOX, and bla_DHA families as well as the bla_KPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla_CTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla_SHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC₉₀ = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.Throughout the 60-year clinical experience with β-lactams and broad-spectrum cephalosporins, bacterial pathogens have kept pace with the introduction of novel agents through the evolution of new β-lactamases that efficiently conform to the new antibacterials (5, 10, 20, 33). One of the most clinically important groups of β-lactamases is the extended-spectrum β-lactamases (ESBLs). Numerous studies have revealed increased mortality and, in general, poorer clinical outcomes for patients infected with bacteria encoding ESBLs (35, 45). According to Bush et al. (11), ESBLs are defined as oxyimino-cephalosporin-hydrolyzing β-lactamases that are inhibited by clavulanic acid.The current rising threat in the clinic is the CTX-M family of ESBLs; these enzymes have appeared in both community and nosocomial settings (3, 12, 38). CTX-M, a class A enzyme, was originally defined by the preferential cleavage of cefotaxime (CTX) versus ceftazidime (CAZ), although several recent derivatives that cleave both agents equivalently have been described (4, 39). In most European countries, Latin America, and East Asia, CTX-M variants have displaced TEM and SHV enzymes as the predominant β-lactamases produced by gram-negative pathogens, especially Klebsiella pneumoniae and Escherichia coli (3, 12, 26). The Lahey website (http://www.lahey.org/Studies/) reports that 82 CTX-M derivatives have been described.TEM-1, the first plasmid-mediated β-lactamase identified in gram-negative organisms, initially described in E. coli in the 1960s (16), is now present in a number of members of the family Enterobacteriaceae, including Klebsiella pneumoniae and Proteus mirabilis, and has spread into additional gram-negative pathogens (5). At last count (http://www.lahey.org/Studies/), more than 160 TEM derivatives have been reported, although not all have an expanded spectrum. In a similar fashion, SHV-type ESBLs have evolved from the SHV-1 enzyme common to K. pneumoniae. They are found on both the chromosome and plasmids and have migrated into Citrobacter spp., E. coli, Pseudomonas aeruginosa, and other genera. Approximately 112 SHV derivatives have been reported; however, not all of the SHV derivatives described are ESBLs (http://www.lahey.org/Studies/). Lastly, OXA, a diverse family of enzymes belonging to the class D family, was initially characterized by rates of hydrolysis of oxacillin that were faster than the rates of hydrolysis of benzylpenicillin and relatively less sensitivity to inhibition by clavulanic acid (3, 5, 11). Over 130 OXA derivatives have been reported; however, not all of the OXA derivatives described have an extended spectrum (http://www.lahey.org/Studies/).The current study was designed to identify, at the sequence level, the genetic determinants responsible for the ESBL phenotype of E. coli, K. pneumoniae, and P. mirabilis isolates collected during the global tigecycline phase 3 clinical trials. In addition, isolates were screened for several AmpC determinants and the carbapenemase-encoding bla_KPC genes.     

Microbiologic and Clinical Implications of Bacteremia Due to Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae with or without Plasmid-Mediated AmpC β-Lactamase DHA-1

Bacteremias caused by Klebsiella pneumoniae producing extended-spectrum beta-lactamase (ESBL-KP; n = 52) and producing both ESBL and AmpC-type DHA-1 beta-lactamase (ESBL-PMABL-KP; n = 20) were analyzed. Higher MIC₅₀s and MIC₉₀s for carbapenems, ciprofloxacin, and piperacillin-tazobactam were observed with ESBL-PMABL-KP than with ESBL-KP. Patients with oxyimino-β-lactam exposure and high modified Pitt bacteremia scores (HMPBSs) were at higher risk, while those with piperacillin-tazobactam and aminoglycoside exposure were at lower risk for ESBL-KP bacteremia. Patients with fluoroquinolone exposure, diabetes mellitus, and HMPBS were at higher risk, while those with aminoglycoside exposure were at lower risk, for ESBL-PMABL-KP bacteremia.Enterobacteriaceae producing an extended-spectrum β- lactamase (ESBL) or a plasmid-mediated AmpC β-lactamase (PMABL) are resistant to broad-spectrum penicillins and oxyimino-β-lactams but usually remain susceptible to carbapenems (5, 11, 16). Strains producing PMABLs are also resistant to cephamycins. Flomoxef, a cephamycin unique in having a difluoromethylthioacetamido group at position 7, has better in vitro activity against ESBL-producing Enterobacteriaceae than other cephamycins (8) but is hydrolyzed by PMABLs (5, 8, 11).In 2004, a cluster of in nosocomial bloodstream infections (BSIs) caused by flomoxef-resistant ESBL-producing Klebsiella pneumoniae isolates was noted at our hospital. In this work, we investigated those isolates and found that they also produced a PMABL. We also compared the clinical and microbiological features of bacteremic infections caused by ESBL plus PMABL-producing K. pneumoniae (ESBL-PAMBL-KP) with infections caused by K. pneumoniae producing only an ESBL (ESBL-KP) or lacking any of those enzymes.ESBL production in K. pneumoniae was screened and then confirmed as recommended by the CLSI (1). Susceptibility testing was performed using the Etest (AB Biodisk, Solna, Sweden). Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as control strains. The breakpoints for susceptibility were ≤8 mg/liter for flomoxef (4) and according to the CLSI (2) for the other agents. As described previously, we identified the ESBL types using PCR amplification and sequencing for bla_SHV, bla_TEM, and bla_CTX-M (7), detected PMABL genes by use of multiplex PCR (13), and genotyped the K. pneumoniae isolates by use of pulsed-field gel electrophoresis (PFGE) (3). The PFGE profiles were interpreted as previously proposed (18).The studied bacteremic patients belonged in two groups, group 1 (ESBL-KP bacteremia; n = 52) and group 2 (ESBL-PMABL-KP bacteremia; n = 20). A control group of patients with nosocomially acquired cefazolin-susceptible K. pneumoniae bacteremia (n = 100) from the same ward and the same period was included for comparison. Demographic and clinical information for patients of the three groups are reported in Table ​Table1.1. Comparison of contingency data was carried out using the Chi-square test or Fisher''s exact test, while comparison of continuous data was performed using Student''s t test or the Mann-Whitney U test. For comparison to the control group, variables in group 1 and group 2 with P values of <0.1 in univariate analyses were separately entered into a multiple logistic regression model to identify the independent risk(s) for BSIs caused by ESBL-KP and ESBL-PMABL-KP, respectively. All comparisons were performed using SPSS 15 software for Windows (SPSS, Inc., Chicago, IL).

TABLE 1.

Demographic and clinical information of bloodstream infections due to K. pneumoniae^a

Cefotaxime and Amoxicillin-Clavulanate Synergism against Extended-Spectrum-β-Lactamase-Producing Escherichia coli in a Murine Model of Urinary Tract Infection

We investigated the efficacies of cefotaxime (CTX) and amoxicillin (AMX)-clavulanate (CLA) (AMC) against extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli in vitro and in a murine model of urinary tract infection (UTI). MICs, the checkerboard dilution method, and time-kill curves were used to explore the in vitro synergism between cefotaxime and amoxicillin-clavulanate against two isogenic E. coli strains—CFT073-RR and its transconjugant, CFT073-RR Tc bla_CTX-M-15—harboring a bla_CTX-M-15 plasmid and a bla_OXA-1 plasmid. For in vivo experiments, mice were separately infected with each strain and treated with cefotaxime, amoxicillin, and clavulanate, alone or in combination, or imipenem, using therapeutic regimens reproducing time of free-drug concentrations above the MIC (fT≥MIC) values close to that obtained in humans. MICs of amoxicillin, cefotaxime, and imipenem were 4/>1,024, 0.125/1,024, and 0.5/0.5 mg/liter, for CFT073-RR and CFT073-RR Tc bla_CTX-M-15, respectively. The addition of 2 mg/liter of clavulanate (CLA) restored the susceptibility of CFT073-RR Tc bla_CTX-M-15 to CTX (MICs of the CTX-CLA combination, 0.125 mg/liter). The checkerboard dilution method and time-kill curves confirmed an in vitro synergy between amoxicillin-clavulanate and cefotaxime against CFT073-RR Tc bla_CTX-M-15. In vivo, this antibiotic combination was similarly active against both strains and as effective as imipenem. In conclusion, the cefotaxime and amoxicillin-clavulanate combination appear to be an effective, easy, and already available alternative to carbapenems for the treatment of UTI due to CTX-M-producing E. coli strains.     

Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom

Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried bla_CTX-M-15 and bla_TEM-1, while K. pneumoniae subsp. pneumoniae isolates carried bla_SHV-12 and bla_TEM-1. Conjugation experiments demonstrated that bla_CTX-M-15 and bla_TEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with bla_CTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the bla_CTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of bla_CTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.     

In Vitro Activities of 21 Antimicrobial Agents Alone and in Combination with Aminoglycosides or Fluoroquinolones against Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates Causing Bacteremia

We evaluated the in vitro activity of various antimicrobials alone and in combination against 291 extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) isolates causing bacteremia in South Korean hospitals. Ceftazidime, cefepime, and piperacillin-tazobactam in combination with amikacin showed greater activity than found in combination with ciprofloxacin. In settings with a high prevalence of ESBL-producing pathogens, combination aminoglycoside antimicrobial therapy, especially with amikacin, may be considered for empirical therapy against suspected Gram-negative sepsis as a carbapenem-saving strategy.     

Does the Activity of the Combination of Imipenem and Colistin In Vitro Exceed the Problem of Resistance in Metallo-β-Lactamase-Producing Klebsiella pneumoniae Isolates?

Using time-kill methodology, we investigated the interactions of an imipenem-colistin combination against 42 genetically distinct Klebsiella pneumoniae clinical isolates carrying a bla_VIM-1-type gene. Irrespective of the imipenem MIC, the combination was synergistic (50%) or indifferent (50%) against colistin-susceptible strains, while it was antagonistic (55.6%) and rarely synergistic (11%) against non-colistin-susceptible strains (with synergy being observed only against strains with colistin MICs of 3 to 4 μg/ml). The combination showed improved bactericidal activity against isolates susceptible either to both agents or to colistin.During the past decade, VIM metallo-β-lactamases (MBLs) have spread rapidly among Enterobacteriaceae (4). MBL producers commonly exhibit a multiple-drug resistance phenotype as a result of combined chromosomally encoded or plasmid-mediated resistance mechanisms. Frequently, colistin and tigecycline remain the only therapeutic choices. Tigecycline has demonstrated in vitro activity against MBL producers (18), but evidence of in vivo efficacy against a variety of clinical infections (i.e., bacteremia or pneumonia) is still limited. On the other hand, randomized controlled trials supporting the use of colistin as a single-drug regimen, as well as studies on its pharmacokinetic/pharmacodynamic properties, are lacking (11). Recently, the emergence of colistin resistance among Klebsiella pneumoniae isolates further jeopardized the already limited treatment options in the intensive care unit setting (2). For all these reasons, combination therapies are frequently used in clinical practice, especially in hospitals with high rates of infections by MBL producers.(Some of these data were presented at the 45th Infectious Disease Society Annual Meeting, 2007 [15a].)We investigated the in vitro activities of imipenem and colistin alone and in combination against 42 unique clinical isolates of MBL-producing K. pneumoniae isolated in Greek hospitals from February 2004 to September 2006. MICs were determined by Etest (AB Biodisc, Solna, Sweden) and interpreted according to CLSI breakpoints for imipenem (3) and EUCAST breakpoints for colistin (7). The presence of a bla_VIM gene was confirmed by PCR (15). On the basis of PCR-restriction fragment length polymorphism analysis (9), all isolates carried a bla_VIM-1-type gene. Extended-spectrum β-lactamase production was detected with a modified CLSI confirmatory test (8). Genetic relatedness among studied isolates was evaluated with repetitive extragenic palindromic PCR methods (10). Patterns that differed by more than one amplification band were characterized as different. In vitro interactions between imipenem and colistin were tested using time-kill methodology. Antibiotic concentrations used were 10 μg/ml for imipenem (Merck, Rahway, NJ) and 5 μg/ml for colistin sulfate (Sigma, St. Louis, MO) because these concentrations represent the steady state achievable in human serum during treatment (12, 16) and thus are clinically relevant. For susceptible strains, if 4× MIC was not higher than 10 or 5 μg/ml for imipenem or colistin, respectively, this concentration was also tested.Synergy was defined as a ≥2-log₁₀ decrease in CFU/ml between the combination and the most active single agent at the different time points, with the number of surviving organisms in the presence of the combination being ≥2 log₁₀ CFU/ml below the number of organisms in the starting inoculum. Antagonism was defined as a ≥2-log₁₀ increase in CFU/ml between the combination and the most active single agent. All other interactions were characterized as indifferent. Bactericidal activity of single antibiotics or combinations was defined as a ≥3-log₁₀ reduction in the CFU/ml of the initial inoculum after 24 h of incubation (1, 6). The lower limit of detection was 1.6 log₁₀ CFU/ml. For analysis of the results, isolates were classified into four groups according to susceptibility to imipenem and colistin. The chi-square test was used to compare proportions of killing activity or synergy between groups by using Yates continuity correction in two-by-two tables. P values of <0.05 were considered statistically significant.The results are shown in Table ​Table1.1. The imipenem-colistin combination exhibited synergy against 12 of 24 (50%) colistin-susceptible MBL-producing K. pneumoniae isolates tested, but it was antagonistic against 10 of 18 (55.6%) non-colistin-susceptible isolates. Of note, isolates showing colistin MICs of 3 to 4 μg/ml behaved more like colistin-susceptible isolates, since in two of them (50%) a synergistic interaction was noted after exposure to the combination.

TABLE 1.

MICs (μg/ml) of imipenem and colistin against bla_VIM-1-type MBL-producing K. pneumoniae isolates and in vitro interaction of the combination