Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches.

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID® ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-Ent strains (24 Escherichia coli) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool ("native SMS") with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach.

Travelers Can Import Colistin-Resistant Enterobacteriaceae, Including Those Possessing the Plasmid-Mediated mcr-1 Gene.

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp ISApl1-mcr-1-ISApl1 element in an IncHI2 plasmid backbone.

Detection, treatment, and prevention of carbapenemase-producing Enterobacteriaceae: recommendations from an International Working Group.

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has increased during the past 10 years. Its detection is frequently difficult, because they do not always show a minimum inhibitory concentration (MIC) value for carbapenems in the resistance range. Both broth microdilution and agar dilution methods are more sensitive than disk diffusion method, Etest and automated systems. Studies on antimicrobial treatment are based on a limited number of patients; therefore, the optimal treatment is not well established. Combination therapy with two active drugs appears to be more effective than monotherapy. Combination of a carbapenem with another active agent--preferentially an aminoglycoside or colistin--could lower mortality provided that the MIC is ≤4 mg/l and probably ≤8 mg/l, and is administered in a higher-dose/prolonged-infusion regimen. An aggressive infection control and prevention strategy is recommended, including reinforcement of hand hygiene, using contact precautions and early detection of CPE through use of targeted surveillance.

Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy?

Despite many years of clinical experience with cefepime, data regarding the outcome of patients suffering from bloodstream infections (BSIs) due to Enterobacter cloacae (Ecl) are scarce. To address the gap in our knowledge, 57 Ecl responsible for 51 BSIs were analysed implementing phenotypic and molecular methods (microarrays, PCRs for bla and other genes, rep-PCR to analyse clonality). Only two E. cloacae (3.5%) were ESBL-producers, whereas 34 (59.6%) and 18 (31.6%) possessed inducible (Ind-Ecl) or derepressed (Der-Ecl) AmpC enzymes, respectively. All isolates were susceptible to imipenem, meropenem, gentamicin and ciprofloxacin. Der-Ecl were highly resistant to ceftazidime and piperacillin/tazobactam (both MIC90≥256 µg/mL), whereas cefepime retained its activity (MIC90 of 3 µg/mL). rep-PCR indicated that the isolates were sporadic, but Ecl collected from the same patients were indistinguishable. In particular, three BSIs initially due to Ind-Ecl evolved (under ceftriaxone or piperacillin/tazobactam treatment) into Der-Ecl because of mutations or a deletion in ampD or insertion of IS4321 in the promoter. These last two mechanisms have never been described in Ecl. Mortality was higher for BSIs due to Der-Ecl than Ind-Ecl (3.8% vs. 29.4%; P=0.028) and was associated with the Charlson co-morbidity index (P=0.046). Using the following directed treatments, patients with BSI showed a favourable treatment outcome: cefepime (16/18; 88.9%); carbapenems (12/13; 92.3%); ceftriaxone (4/7; 57.1%); piperacillin/tazobactam (5/7; 71.4%); and ciprofloxacin (6/6; 100%). Cefepime represents a safe therapeutic option and an alternative to carbapenems to treat BSIs due to Ecl when the prevalence of ESBL-producers is low.

Evaluation of ceftazidime and NXL104 in two murine models of infection due to KPC-producing Klebsiella pneumoniae.

We evaluated the efficacy of NXL104, a novel ß-lactamase inhibitor, in combination with ceftazidime (CAZ) in two murine infection models (septicemia and thigh infection). We chose two KPC-producing Klebsiella pneumoniae strains (VA-361 and VA-406) showing MICs of CAZ of ≥256 µg/ml. Septicemia was induced by the intraperitoneal injection of KPC-producing K. pneumoniae followed 30 min later by a single subcutaneous treatment with CAZ alone or CAZ-NXL104 in ratios of 2:1, 4:1, 8:1, and 16:1. In this model, the median effective doses for 50% (ED(50)) of the animals for CAZ alone versus VA-361 and VA-406 were 1,578 and 709 mg/kg of body weight, respectively. When combined with NXL104 at 2:1, 4:1, 8:1, and 16:1 ratios, the CAZ ED(50)s for VA-361 and VA-406 were reduced to 8.1 and 3.5 mg/kg, 15.1 and 3.8 mg/kg, 16.9 and 7.2 mg/kg, and 29.5 and 12.1 mg/kg, respectively. For thigh infection, neutropenia was induced by the intraperitoneal injection of cyclophosphamide at days -4 and -1 preinfection. Infection was established by the intramuscular injection of KPC-producing K. pneumoniae into the right thigh. Mice were treated 1.5 h postinfection with either CAZ alone or CAZ-NXL104 at constant ratios of 4:1. When thighs were removed at 24 h postinfection, a >2-log CFU reduction was observed for mice treated with CAZ-NXL104 at doses of ≥128:32 mg/kg. In contrast, CAZ doses of ≥1,024 mg/kg were unable to reduce the numbers of CFU. Despite resistance to CAZ and possessing a complex ß-lactamase background, NXL104 combined with CAZ proved to be very effective in murine models of infection due to contemporary highly resistant KPC-producing K. pneumoniae isolates.

Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum beta-lactamases.

Bloodstream infection (BSI) due to Proteus mirabilis strains is a relatively uncommon clinical entity, and its significance has received little attention. This study was initiated to evaluate risk factors and treatment outcome of BSI episodes due to P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). Twenty-five BSI episodes caused by P. mirabilis occurred at our hospital (Ospedale di Circolo e Fondazione Macchi, Varese, Italy) over a 7.5-year period. Phenotypic and molecular methods were used to assess ESBL production. Clinical records of BSI patients were examined retrospectively. Demographic data, underlying diseases (according to McCabe and Jackson classification and Charlson weighted index), risk factors, and treatment outcome were investigated by comparing cases due to ESBL-positive strains to cases due to ESBL-negative strains. Eleven isolates were found to express ESBLs (TEM-52 or TEM-92). The remaining 14 isolates were ESBL negative and were uniformly susceptible to extended-spectrum cephalosporins and monobactams. Comparison of the two groups showed that previous hospitalization in a nursing home (P = 0.04) and use of bladder catheter (P = 0.01) were significant risk factors for infections due to ESBL-positive strains. In addition, cases due to ESBL-positive strains showed a significantly higher mortality attributable to BSI (P = 0.04). BSI cases due to ESBL-negative isolates uniformly responded to therapy, whereas 5/11 cases due to ESBL-positive isolates failed to respond (P < 0.01). Use of carbapenems was associated with complete response independently of ESBL production. Therapeutic failure and mortality may occur in BSI episodes caused by ESBL-positive P. mirabilis isolates. Thus, recognition of ESBL-positive strains appears to be critical for the clinical management of patients with systemic P. mirabilis infections.

Bacteremia due to Klebsiella pneumoniae isolates producing the TEM-52 extended-spectrum beta-lactamase: treatment outcome of patients receiving imipenem or ciprofloxacin.

The treatment outcome of 35 cases of bacteremia due to Klebsiella pneumoniae isolates producing TEM-52 extended-spectrum beta-lactamase was studied. Twenty-eight cases, classified as "nonfatal disease" using the McCabe and Jackson classification, were investigated with regard to ciprofloxacin and imipenem response. Because ciprofloxacin was active in vitro against 21 of 28 isolates, only the treatment outcome of the ciprofloxacin-susceptible subgroup was evaluated. Eight of 10 cases occurred in patients who experienced a complete response to imipenem; 2 of 10 failed to respond. In contrast, only 2 of 7 cases had a partial response to ciprofloxacin, and, in 5 of 7 cases, the treatment failed. Statistical analysis revealed a significant difference in the treatment outcome of the 2 groups (P=.03). Because the isolates had minimum inhibitory concentrations of ciprofloxacin close to the susceptibility breakpoint, treatment failure could be ascribed to the inability of the drug to reach therapeutic concentrations at infected sites.