Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands.

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1-pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1-pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1-pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1-pST12 plasmids revealed a low number of SNP differences (range of 0-9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1-pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

Development of amoxicillin resistance in Escherichia coli after exposure to remnants of a non-related phagemid-containing E. coli: an exploratory study.

OBJECTIVE: To determine the effect of exposure to remnants of a phagemid-containing E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates. METHODS: An in vitro model was developed in which a clinical E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development. RESULTS: In 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only (risk difference: 10.0%; 95% CI 5.4-14.6%)) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 (risk difference: 10.5%; 95% CI 6.1-14.9%). In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction. CONCLUSION: Exposure of an E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC ß-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization.

OBJECTIVES: AmpC-ß-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common ß-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. METHODS: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. RESULTS: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2. CONCLUSIONS: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.

Contact precautions in single-bed or multiple-bed rooms for patients with extended-spectrum ß-lactamase-producing Enterobacteriaceae in Dutch hospitals: a cluster-randomised, crossover, non-inferiority study.

BACKGROUND: Use of single-bed rooms for control of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is under debate; the added value when applying contact precautions has not been shown. We aimed to assess whether an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae. METHODS: We did a cluster-randomised, crossover, non-inferiority study on medical and surgical wards of 16 Dutch hospitals. During two consecutive study periods, either contact precautions in a single-bed room or contact precautions in a multiple-bed room were applied as the preferred isolation strategy for patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample (index patients). Eligible index patients were aged 18 years or older, had no strict indication for barrier precautions in a single-bed room, had a culture result reported within 7 days of culture and before discharge, and had no wardmate known to be colonised or infected with an ESBL-producing Enterobacteriaceae isolate of the same bacterial species with a similar antibiogram. Hospitals were randomly assigned in a 1:1 ratio by computer to one of two sequences of isolation strategies, stratified by university or non-university hospital. Allocation was masked for laboratory technicians who assessed the outcomes but not for patients, treating doctors, and infection-control practitioners enrolling index patients. The primary outcome was transmission of ESBL-producing Enterobacteriaceae to wardmates, which was defined as rectal carriage of an ESBL-producing Enterobacteriaceae isolate that was clonally related to the index patient's isolate in at least one wardmate. The primary analysis was done in the per-protocol population, which included patients who were adherent to the assigned room type. A 10% non-inferiority margin for the risk difference was used to assess non-inferiority. This study is registered with Nederlands Trialregister, NTR2799. FINDINGS: 16 hospitals were randomised, eight to each sequence of isolation strategies. All hospitals randomised to the sequence single-bed room then multiple-bed room and five of eight hospitals randomised to the sequence multiple-bed room then single-bed room completed both study periods and were analysed. From April 24, 2011, to Feb 27, 2014, 1652 index patients and 12 875 wardmates were assessed for eligibility. Of those, 693 index patients and 9527 wardmates were enrolled and 463 index patients and 7093 wardmates were included in the per-protocol population. Transmission of ESBL-producing Enterobacteriaceae to at least one wardmate was identified for 11 (4%) of 275 index patients during the single-bed room strategy period and for 14 (7%) of 188 index patients during the multiple-bed room strategy period (crude risk difference 3·4%, 90% CI -0·3 to 7·1). INTERPRETATION: For patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample, an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae. Non-inferiority of the multiple-bed room strategy might change the current single-bed room preference for isolation of patients with ESBL-producing Enterobacteriaceae and, thus, broaden infection-control options for ESBL-producing Enterobacteriaceae in daily clinical practice. FUNDING: Netherlands Organisation for Health Research and Development.

Evaluation of an in vitro model with a novel statistical approach to measure differences in bacterial survival of extended-spectrum ß-lactamase-producing Escherichia coli on an inanimate surface.

Background: The role of environmental contamination in the transmission of Enterobacteriaceae is increasingly recognized. However, factors influencing the duration of survival in the environment have not yet been extensively studied. In this study, we developed and evaluated an in vitro model with a novel statistical approach to accurately measure differences in bacterial survival, that can be used to model the effects of multiple factors/conditions in future experiments. Methods: Two extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) isolates were used for this in vitro experiment: a CTX-M-15-producing E. coli sequence type (ST) 131 and a CTX-M-1-producing E. coli ST10 isolate. Each strain was 1:1 diluted in sterile water, sterile saline or sheep blood. Cover glasses (18 × 18 mm) were inoculated with the dilution and subsequently kept at room temperature. Bacterial survival on the glasses was determined hourly during the first day, once daily during the following 6 days, and from day 7 on, once weekly up to 100 days. The experiment was repeated six times for each strain, per suspension fluid. Results: Viable bacteria could be detected up to 70 days. A biphasic survival curve for all suspension fluids was observed, whereby there was a rapid decrease in the number of viable bacteria in the first 7 h, followed by a much slower decrease in the subsequent days. Conclusions: We found a difference in survival probability between E. coli ST10 and ST131, with a higher proportion of viable bacteria remaining after 7 h for ST131, particularly in sheep blood.

Decreasing prevalence of contamination with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in retail chicken meat in the Netherlands.

Retail chicken meat is a potential source of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). In the past decade, vast national efforts were undertaken to decrease the antibiotic use in the veterinary sector, resulting in a 58% decrease in antibiotic sales in the sector between 2009 and 2014. This decrease in antibiotic use was followed by a decrease in ESBL-E prevalence in broilers. The current study investigates the prevalence of contamination with ESBL-E in retail chicken meat purchased in the Netherlands between December 2013 and August 2015. It looks at associations between the prevalence of contamination with ESBL-E and sample characteristics such as method of farming (free-range or conventional), supermarket chain of purchase and year of purchase. In the current study, 352 chicken meat samples were investigated for the presence of ESBL-E using selective culture methods. Six samples were excluded due to missing isolates or problems obtaining a good quality sequence leaving 346 samples for further analyses. Of these 346 samples, 188 (54.3%) were positive for ESBL-E, yielding 216 ESBL-E isolates (Escherichia coli (n = 204), Klebsiella pneumoniae (n = 11) and Escherichia fergusonii (n = 1)). All ESBL-E isolates were analysed using whole-genome sequencing. The prevalence of contamination with ESBL-E in retail chicken meat decreased from 68.3% in 2014 to 44.6% in 2015, absolute risk difference 23.7% (95% confidence interval (CI): 12.6% - 34.1%). The ESBL-E prevalence was lower in free-range chicken meat (36.4%) compared with conventional chicken meat (61.5%), absolute risk difference 25.2% (95% CI: 12.9% - 36.5%). The prevalence of contamination with ESBL-E varied between supermarket chains, the highest prevalence of contamination was found in supermarket chain 4 (76.5%) and the lowest in supermarket chain 1 (37.8%). Pairwise isolate comparisons using whole-genome multilocus sequence typing (wgMLST) showed that clustering of isolates occurs more frequently within supermarket chains than between supermarket chains. In conclusion, the prevalence of contamination with ESBL-E in retail chicken in the Netherlands decreased over time; nevertheless, it remains substantial and as such a potential source for ESBL-E in humans.

Decline in AmpC ß-lactamase-producing Escherichia coli in a Dutch teaching hospital (2013-2016).

OBJECTIVE: The objective of this study is to determine the prevalence of rectal carriage of plasmid- and chromosome-encoded AmpC ß-lactamase-producing Escherichia coli and Klebsiella spp. in patients in a Dutch teaching hospital between 2013 and 2016. METHODS: Between 2013 and 2016, hospital-wide yearly prevalence surveys were performed to determine the prevalence of AmpC ß-lactamase-producing E. coli and Klebsiella spp. rectal carriage. Rectal swabs were taken and cultured using an enrichment broth and selective agar plates. All E. coli and Klebsiella spp. isolates were screened for production of AmpC ß-lactamase using phenotypic confirmation tests and for the presence of plasmid-encoded AmpC (pAmpC) genes. E. coli isolates were screened for chromosome-encoded AmpC (cAmpC) promoter/attenuator alterations. RESULTS: Fifty (2.4%) of 2,126 evaluable patients were identified as rectal carrier of AmpC ß-lactamase-producing E. coli. No carriage of AmpC ß-lactamase producing Klebsiella spp. was found. Nineteen (0.9%) patients harboured isolates with pAmpC genes and 30 (1,4%) patients harboured isolates with cAmpC promoter/attenuator alterations associated with AmpC ß-lactamase overproduction. For one isolate, no pAmpC genes or cAmpC promotor/attenuator alterations could be identified. During the study period, a statistically significant decline in the prevalence of rectal carriage with E. coli with cAmpC promotor/attenuator alterations was found (p = 0.012). The prevalence of pAmpC remained stable over the years. CONCLUSIONS: The prevalence of rectal carriage of AmpC-producing E. coli and Klebsiella spp. in patients in Dutch hospitals is low and a declining trend was observed for E. coli with cAmpC promotor/attenuator alterations.

Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) isolated from bean sprouts in the Netherlands.

Community-acquired carriage and infections due to extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) are increasing worldwide, resulting in increased morbidity, mortality and healthcare costs. The origins of community-acquired ESBL-E carriage and infections remain unclear. Bean sprouts are a potential source of Enterobacteriaceae for the community, as illustrated by outbreaks of pathogenic Enterobacteriaceae in the past. The current study focuses on contamination of retail bean sprouts with ESBL-E in the Netherlands. Of 131 bean sprout samples purchased between 2013 and 2016, 25 (19%) were contaminated with ESBL-E. The detected isolates were almost exclusively Klebsiella spp. and co-resistance to other antibiotics was observed frequently. Over time there was substantial genetic diversity between isolates. On the other hand, isolates from samples closely matched in time were frequently clonally related, indicative of batch contamination. Remarkably, no Escherichia coli was found. In conclusion, bean sprouts frequently harbor ESBL-E, which is a potential source for consumers.

High prevalence of the mcr-1 gene in retail chicken meat in the Netherlands in 2015.

Recently, plasmid-mediated colistin resistance was reported in humans, animals and food. We studied the presence of mcr-1 and mcr-2 in Dutch retail chicken meat. The prevalence of mcr-1 was 24,8% (53/214), whereas mcr-2 was not found. The presence of mcr-1-positive Enterobacteriaceae was confirmed by culture in 34/53 samples (64,2%). The prevalence depended on the supermarket chain and was lower in free-range chicken samples. The unexpected high prevalence of mcr-1 in food is cause for concern.

Prolonged colonisation with Escherichia coli O25:ST131 versus other extended-spectrum beta-lactamase-producing E. coli in a long-term care facility with high endemic level of rectal colonisation, the Netherlands, 2013 to 2014.

The extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clone ST131 (ESBL-ST131) has spread in healthcare settings worldwide. The reasons for its successful spread are unknown, but might include more effective transmission and/or longer persistence. We evaluated the colonisation dynamics of ESBL-producing E. coli (ESBL-EC), including ESBL-ST131, in a long-term care facility (LTCF) with an unusually high prevalence of rectal ESBL-EC colonisation. During a 14-month period, rectal or faecal samples were obtained from 296 residents during six repetitive prevalence surveys, using ESBL-selective culture. Transmission rates, reproduction numbers, and durations of colonisation were compared for ESBL-ST131 vs other ESBL-EC. Furthermore, the likely time required for ESBL-ST131 to disappear from the LTCF was estimated. Over time, the endemic level of ESBL-ST131 remained elevated whereas other ESBL-EC returned to low-level prevalence, despite comparable transmission rates. Survival analysis showed a half-life of 13 months for ESBL-ST131 carriage, vs two to three months for other ESBL-EC (p < 0.001). Per-admission reproduction numbers were 0.66 for ESBL-ST131 vs 0.56 for other ESBL-EC, predicting a mean time of three to four years for ESBL-ST131 to disappear from the LTCF under current conditions. Transmission rates were comparable for ESBL-ST131 vs other ESBL-EC. Prolonged rectal carriage explained the persistence of ESBL-ST131 in the LTCF.

Retrospective identification of a previously undetected clinical case of OXA-48-producing K. pneumoniae and E. coli: the importance of adequate detection guidelines.

INTRODUCTION: The laboratory detection of OXA-48-carbapenemase-producing Enterobacteriaceae is difficult, as minimum inhibition concentrations for carbapenems are often below the clinical breakpoint. In 2011, the Dutch national guideline for the detection of highly resistant micro-organisms was issued, which includes recommendations on the use of carbapenem screening breakpoints for the detection of carbapenemase-producing Enterobacteriaceae. MATERIALS AND METHODS: During a validation study of the Check-MDR CT103 microarray (Check-Points, Wageningen, The Netherlands) in 2013, an OXA-48-like carbapenemase gen was identified in two isolates that were previously obtained from a patient with non-Hodgkin lymphoma in 2007. Whole-genome sequencing (WGS) and subsequent BLAST Ringe Image Generator (BRIG) analysis were performed to establish the presence of OXA-48 carbapenemase encoding plasmids and their similarity. RESULTS: This case report describes the first documented OXA-48-producing Klebsiella pneumonia (ST648) and Escherichia coli (ST866) in the Netherlands. A similar IncL/M plasmid was identified in both strains, suggesting within-patient horizontal transfer. CONCLUSION: This case illustrates that OXA-48-carbapenemase-producing Enterobacteriaceae can be unnoticed without adequate laboratory detection procedures. Our observation stresses the importance of uniform and adequate laboratory methods for the timely and accurate detection of important antimicrobial resistance.

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.

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients.

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield.

Extensive dissemination of extended spectrum ß-lactamase-producing Enterobacteriaceae in a Dutch nursing home.

OBJECTIVE: Risk factors for rectal carriage of ESBL-E and transmission were investigated in an outbreak of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E). DESIGN: Rectal carriage of ESBL-E was determined in a cross-sectional survey by culture of perianal swabs or fecal samples. Both phenotypical and genotypical methods were used to detect the production of ESBL. Nosocomial transmission was defined as the presence of genotypically related strains in ≥2 residents within the NH. Patient characteristics and variables in infection control practices were registered to investigate risk factors for transmission. SETTING: A nursing home (NH) in the southern Netherlands. PARTICIPANTS: Of 189 residents, 160 residents (84.7%) were screened for ESBL-E carriage. Of these 160 residents, 33 (20.6%) were ESBL-E positive. ESBL carriage rates varied substantially between wards (range, 0-47%). Four different ESBL-E clusters were observed. A bla CTX-M1-15 positive E. coli ST131 constituted the largest cluster (n=21) and was found in multiple wards (n=7). RESULTS: Our investigation revealed extensive clonal dissemination of bla CTX-M1-15-positive E. coli ST131 in a nursing home. Unexplained differences in ESBL prevalence were detected among the wards. CONCLUSIONS: As NHs constitute potential sources of multidrug-resistant bacteria, it is important to gain a better understanding of the risks factors and routes of transmission of ESBL-E.

Methicillin-resistant Staphylococcus aureus in meat products, the Netherlands.

A new methicillin-resistant Staphylococcus aureus (MRSA) clone related to pig and cattle farming was detected in the Netherlands. We investigated the extent of S. aureus presence in meat and found 36 S. aureus strains in 79 samples. Two strains were MRSA; 1 was multilocus sequence type 398, the clone related to farming.

Pseudo-outbreak of hepatitis B virus infection associated with contamination of a semiautomatic cap remover.

A pseudo-outbreak of hepatitis B virus caused by cross-contamination from a semiautomatic cap remover for blood collection tubes is reported. The source of the outbreak was elucidated by using basic epidemiological methods. Laboratories should always be critical about their results in order to identify contamination problems.