Ina Willemsen

Extended-Spectrum β-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands

We determined the prevalence and characteristics of extended-spectrum β-lactamase (ESBL) genes of Enterobacteriaceae in retail chicken meat and humans in the Netherlands. Raw meat samples were obtained, and simultaneous cross-sectional surveys of fecal carriage were performed in 4 hospitals in the same area. Human blood cultures from these hospitals that contained ESBL genes were included. A high prevalence of ESBL genes was found in chicken meat (79.8%). Genetic analysis showed that the predominant ESBL genes in chicken meat and human rectal swab specimens were identical. These genes were also frequently found in human blood culture isolates. Typing results of Escherichia coli strains showed a high degree of similarity with strains from meat and humans. These findings suggest that the abundant presence of ESBL genes in the food chain may have a profound effect on future treatment options for a wide range of infections caused by gram-negative bacteria.

Extended-Spectrum β-Lactamase–Producing Escherichia coli From Retail Chicken Meat and Humans: Comparison of Strains, Plasmids, Resistance Genes, and Virulence Factors

BACKGROUND The worldwide prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly both in hospitals and in the community. A connection between ESBL-producing bacteria in food animals, retail meat, and humans has been suggested. We previously reported on the genetic composition of a collection of ESBL-producing Escherichia coli (ESBL-EC) from chicken meat and humans from a restricted geographic area. Now, we have extended the analysis with plasmid replicons, virulence factors, and highly discriminatory genomic profiling methods. METHODS One hundred forty-five ESBL-EC isolates from retail chicken meat, human rectal carriers, and blood cultures were analyzed using multilocus sequence typing, phylotyping, ESBL genes, plasmid replicons, virulence genes, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). RESULTS Three source groups overlapped substantially when their genetic composition was compared. A combined analysis using all variables yielded the highest resolution (Wilks lambda [Λ]: 0.08). Still, a prediction model based on the combined data classified 40% of the human isolates as chicken meat isolates. AFLP and PFGE showed that the isolates from humans and chicken meat could not be segregated and identified 1 perfect match between humans and chicken meat. CONCLUSIONS We found significant genetic similarities among ESBL-EC isolates from chicken meat and humans according to mobile resistance elements, virulence genes, and genomic backbone. Therefore, chicken meat is a likely contributor to the recent emergence of ESBL-EC in human infections in the study region. This raises serious food safety questions regarding the abundant presence of ESBL-EC in chicken meat.

Appropriateness of Antimicrobial Therapy Measured by Repeated Prevalence Surveys

ABSTRACT Prudent use of antibiotics is mandatory to control antibiotic resistance. The objective of this study was to determine if prevalence surveys are useful tools to determine the appropriateness of antimicrobial therapy (AMT) and determinants of inappropriate AMT. The study was performed in a 1,350-bed teaching hospital including all medical specialities. Six consecutive 1-day prevalence surveys of in-patients were performed twice yearly from 2001 to 2004. Data on the demographics, infections, and AMT were gathered. The appropriateness of AMT was assessed according to a standardized algorithm based on the local AMT prescription guidelines. On average, 684 patients were included in each survey (total, 4,105). The use of AMT as determined in the prevalence survey corresponded to the annual data from the pharmacy department. Nine hundred thirty-eight (22.9%) of the patients received AMT, and in 351 (37.4%) of these patients AMT was inappropriate. Only 25 (0.6%) patients did not receive AMT, although it was indicated. After multivariate analysis, the use of quinolones was the only statistically significant variable associated with inappropriate use. Prevalence surveys proved to be useful tools to judge the appropriateness of AMT and to identify determinants of inappropriate use. This study shows that in a setting with a low use of AMT, there are few patients who inadvertently do not receive AMT. On the other hand, a substantial number of the patients are treated inappropriate.

Correlation between antibiotic use and resistance in a hospital: Temporary and ward-specific observations

Objectives:The objectives of this study were to determine (1) the increase in antimicrobial resistance to frequently used antibiotics in the hospital setting over time and (2) the correlation between the amount of use of an antibiotic in a specific medical specialty and the observed resistance to that antibiotic in that specialty.Method:The total use of antibiotics and the use of ciprofloxacin (CIP), co-amoxicillin + clavulanic acid (AMCL) and firstand second-generation cephalosporins (CEF), respectively, in individual medical specialties were measured between 2001 and 2006 by means of prevalence surveys (two per year). The antimicrobial susceptibility patterns among E. coli isolated from hospitalized patients between 2003 and 2006 were obtained from the Laboratory Information System. Trends over time and correlation between use and resistance were calculated.Results:6,639 patients were included in the prevalence surveys, of whom 3.0% (195) were treated with CIP, 9.7% (642) with AMCL, and 3.5% (232) with CEF. 4,790 E. coli isolates were obtained from hospitalized patients. Resistance to all antibiotics significantly increased over time, with the regression line showing that the strongest increase in resistance was for CIP (2.6% per year). There were large variations in antimicrobial use between various medical specialties. A significant correlation was found between the ward-specific prevalence of use and the percentage of resistance for CIP (R = 0.81, p < 0.001) and AMCL (R = 0.82, p = 0.003).Conclusion:At the level of individual medical specialties within one hospital, a higher prevalence of antimicrobial use among patients was associated with a significantly higher observed antimicrobial resistance. The use of CIP was associated with a stronger increase in resistance than the use of beta-lactams.

Laboratory Detection of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae: Evaluation of Two Screening Agar Plates and Two Confirmation Techniques

ABSTRACT The worldwide prevalence of extended-spectrum-beta-lactamase-producing ESBL-producing Enterobacteriaceae (ESBL-E) is increasing, making the need for optimized detection techniques more urgent. In this study we investigated the performance of two ESBL-E screening and two ESBL-E confirmation techniques. In accordance with the Dutch national guidelines (www.wip.nl), a collection of 642 highly resistant Enterobacteriaceae strains, as identified by Vitek2, was used to test the performances of two screening techniques (EbSA ESBL agar plate and ChromID ESBL agar plate) and of two confirmation techniques (MIC-strip ESBL and Vitek2 ESBL test panel). The individual test results were compared by using Etest, followed by a combination disk test if Etest results were inconclusive. Among group 1 isolates (Escherichia coli, Klebsiella spp., Proteus spp., Salmonella spp., and Shigella spp.) 291 (57.6%) were ESBL-E, versus 65 (47.4%) in group 2 (Enterobacter spp., Citrobacter spp., Morganella morganii, Serratia spp., and Providencia spp.). The sensitivities of all four tests for group 1 were comparable (EbSA, 96.6%; ChromID, 97.3%; MIC-strip, 99.6%; and Vitek2, 95.1%). The specificities of the EbSA and ChromID were the same (93.9%). However, the confirmation techniques produced many inconclusive test results, which reduces the applicability in routine laboratories. Only the two screening agar plates were validated for ESBL testing of group 2 microorganisms. They showed comparable sensitivities; however, the EbSA screening agar plate had a significantly higher specificity (78.6% versus 44.3%). In conclusion the screening agar plates performed better than the two confirmation techniques. The EbSA agar plate had the best overall performance.

Trends in Extended Spectrum Beta-Lactamase (ESBL) Producing Enterobacteriaceae and ESBL Genes in a Dutch Teaching Hospital, Measured in 5 Yearly Point Prevalence Surveys (2010-2014)

This paper describes the trends in prevalence of ESBL producing Enterobacteriaceae (ESBL-E) and ESBL genes, measured in five consecutive yearly Point Prevalence Surveys (PPS). All patients present in the hospital and in a day-care clinic (including patients on dialysis) on the day of the survey, were screened for perianal ESBL-E carriage. Perianal swabs were taken and cultured using an enrichment broth and a selective agar plate. Both phenotypic and genotypic methods were used to detect the production of ESBL, presence of ESBL-genes and clonal relatedness. Out of 2,695 patients, 135 (5.0%) were tested ESBL-E positive. The overall ESBL-E prevalence was stable over the years. Overall 5.2% of all ESBL-E were acquired by nosocomial transmission. A relative decrease of CTX-M-1-1-like ESBL genes (from 44 to 25%, p = 0.026) was observed, possibly related to the strong (>60%) decrease in antibiotic use in livestock in our country during the same period.

Improving Quinolone Use in Hospitals by Using a Bundle of Interventions in an Interrupted Time Series Analysis

ABSTRACT The objectives of the present study were to determine the effects of multiple targeted interventions on the level of use of quinolones and the observed rates of resistance to quinolones in Escherichia coli isolates from hospitalized patients. A bundle consisting of four interventions to improve the use of quinolones was implemented. The outcome was measured from the monthly levels of use of intravenous (i.v.) and oral quinolones and the susceptibility patterns for E. coli isolates from hospitalized patients. Statistical analyses were performed using segmented regression analysis and segmented Poisson regression models. Before the bundle was implemented, the annual use of quinolones was 2.7 defined daily doses (DDDs)/100 patient days. After the interventions, in 2007, this was reduced to 1.7 DDDs/100 patient days. The first intervention, a switch from i.v. to oral medication, was associated with a stepwise reduction in i.v. quinolone use of 71 prescribed daily doses (PDDs) per month (95% confidence interval [CI] = 47 to 95 PDDs/month, P < 0.001). Intervention 2, introduction of a new antibiotic guideline and education program, was associated with a stepwise reduction in the overall use of quinolones (reduction, 107 PDDs/month [95% CI = 58 to 156 PDDs/month). Before the interventions the quinolone resistance rate was increasing, on average, by 4.6% (95% CI = 2.6 to 6.1%) per year. This increase leveled off, which was associated with intervention 2 and intervention 4, active monitoring of prescriptions and feedback. Trends in resistance to other antimicrobial agents did not change. This study showed that the hospital-wide use of quinolones can be significantly reduced by an active policy consisting of multiple interventions. There was also a stepwise reduction in the rate of quinolone resistance associated with the bundle of interventions.

Highly resistant microorganisms in a teaching hospital: the role of horizontal spread in a setting of endemicity.

OBJECTIVE To determine the incidence density of highly resistant organisms (HROs) and the relative contribution of horizontal spread in a setting of endemicity. METHODS Prospective surveillance was performed among hospitalized patients during an 18-month period. Enterobacteriaceae, nonfermentative gram-negative bacilli, Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecium--all considered highly resistant, according to Dutch guidelines--were included. Epidemiological linkage and nosocomial transmission were determined on the basis of molecular typing and hospital admission data. RESULTS From 119 patients, we recovered a total of 170 unique HRO isolates, as follows: Escherichia coli, 96 isolates; Klebsiella species, 11 isolates; Enterobacter species, 8 isolates; Proteus species, 9 isolates; Citrobacter species, 5 isolates; Pseudomonas species, 5 isolates; Acinetobacter species, 3 isolates; Morganella species, 2 isolates; Salmonella species, 1 isolate; Serratia species, 1 isolate; S. pneumoniae, 20 isolates; and S. aureus, 9 isolates. No vancomycin-resistant E. faecium was found. The incidence density was 4.3 HRO isolates per 10,000 patient-days. The majority of HRO isolates were unique, and nosocomial transmission was observed 4 times for highly resistant gram-negative bacilli (case reproduction rate, 0.05) and 4 times for penicillin-nonsusceptible S. pneumoniae (case reproduction rate, 0.29). A stay on the intensive care unit was the main determinant for the recovery of an HRO. CONCLUSION Nosocomial transmission of HROs was observed 8 times during the 18-month period. The intensive care unit was identified as the main reservoir of horizontal spread of HROs. This study shows that nosocomial transmission of HROs is largely preventable using transmission precautions.

Detection and Occurrence of Plasmid-Mediated AmpC in Highly Resistant Gram-Negative Rods

Objectives The aim of this study was to compare the current screening methods and to evaluate confirmation tests for phenotypic plasmidal AmpC (pAmpC) detection. Methods For this evaluation we used 503 Enterobacteriaceae from 18 Dutch hospitals and 21 isolates previously confirmed to be pAmpC positive. All isolates were divided into three groups: isolates with 1) reduced susceptibility to ceftazidime and/or cefotaxime; 2) reduced susceptibility to cefoxitin; 3) reduced susceptibility to ceftazidime and/or cefotaxime combined with reduced susceptibility to cefoxitin. Two disk-based tests, with cloxacillin or boronic acid as inhibitor, and Etest with cefotetan-cefotetan/cloxacillin were used for phenotypic AmpC confirmation. Finally, presence of pAmpC genes was tested by multiplex and singleplex PCR. Results We identified 13 pAmpC producing Enterobacteriaceae isolates among the 503 isolates (2.6%): 9 CMY-2, 3 DHA-1 and 1 ACC-1 type in E. coli isolates. The sensitivity and specificity of reduced susceptibility to ceftazidime and/or cefotaxime in combination with cefoxitin was 97% (33/34) and 90% (289/322) respectively. The disk-based test with cloxacillin showed the best performance as phenotypic confirmation method for AmpC production. Conclusions For routine phenotypic detection of pAmpC the screening for reduced susceptibility to third generation cephalosporins combined with reduced susceptibility to cefoxitin is recommended. Confirmation via a combination disk diffusion test using cloxacillin is the best phenotypic option. The prevalence found is worrisome, since, due to their plasmidal location, pAmpC genes may spread further and increase in prevalence.

New Diagnostic Microarray (Check-KPC ESBL) for Detection and Identification of Extended-Spectrum Beta-Lactamases in Highly Resistant Enterobacteriaceae

ABSTRACT The performance of a microarray for the detection of extended-spectrum beta-lactamases was determined on a collection of 638 highly resistant members of the family Enterobacteriaceae collected from patients in 18 hospitals in The Netherlands. The microarray had a significantly higher specificity than the phenotypic assays. It also detects carbapenemases and characterizes the resistance genes, providing epidemiological insight.