Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands.

OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.

Characterization of mobile genetic elements in multidrug-resistant Bacteroides fragilis isolates from different hospitals in the Netherlands.

OBJECTIVES: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. METHODS: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). RESULTS: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. CONCLUSIONS: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.

The Microbiome in Bronchial Biopsies from Smokers and Ex-Smokers with Stable COPD - A Metatranscriptomic Approach.

Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n = 5) and ex-smokers (n = 6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways.

Three metronidazole-resistant Prevotella bivia strains harbour a mobile element, encoding a novel nim gene, nimK, and an efflux small MDR transporter.

Objectives: In this study we assess the antibiotic resistance genes in three metronidazole-resistant Prevotella bivia clinical isolates. Methods: Strains were whole-genome sequenced. De novo assembly was performed and genes were annotated in RAST. Manual adjustments were made, when required, to the annotation and length of the genes. Results: In all three strains a novel nim gene, nimK, was encountered located on a mobile genetic element (MGE). The nimK gene was associated with an IS1380 family transposase. On the same MGE, genes encoding an efflux small MDR (SMR) transporter were present and were associated with a crp/fnr regulator. Conclusions: This is the first description of the presence of a novel nim gene in metronidazole-resistant P. bivia clinical isolates. This gene is co-located with an efflux SMR transporter on an MGE, which has been named Tn6456 (MG827401). The identification of these resistance genes on an MGE is worrisome, since this indicates the horizontal gene transfer of antibiotic and/or biocide resistance from one strain to the other.

Elucidating vancomycin-resistant Enterococcus faecium outbreaks: the role of clonal spread and movement of mobile genetic elements.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons. Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks. Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses. Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon. Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.

Extensive colonization with carbapenemase-producing microorganisms in Romanian burn patients: infectious consequences from the Colectiv fire disaster.

Health care of severe burn patients is highly specialized and may require international patient transfer. Burn patients have an increased risk of developing infections. Patients that have been hospitalized in countries where carbapenemase-producing microorganisms (CPMO) are endemic may develop infections that are difficult to treat. In addition, there is a risk on outbreaks with CPMOs in burn centers. This study underlines that burn patients may extensively be colonized with CPMOs, and it provides best practice recommendations regarding clinical microbiology and infection control. We evaluated CPMO-carriage and wound colonization in a burn patient initially treated in Romania, and transported to the Netherlands. The sequence types and acquired beta-lactamase genes of highly-resistant microorganisms were derived from next generation sequencing data. Next, we searched literature for reports on CPMOs in burn patients. Five different carbapenemase-producing isolates were cultured: two unrelated OXA-48-producing Klebsiella pneumoniae isolates, OXA-23-producing Acinetobacter baumanii, OXA-48-producing Enterobacter cloacae, and NDM-1-producing Providencia stuartii. Also, multi-drug resistant Pseudomonas aeruginosa isolates were detected. Among the sampling sites, there was high variety in CPMOs. We found 46 reports on CPMOs in burn patients. We listed the epidemiology of CPMOs by country of initial treatment, and summarized recommendations for care of these patients based on these reports and our study.

Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.

Enterovirus and parechovirus infection in children: a brief overview.

UNLABELLED: Enterovirus and parechovirus are a frequent cause of infection in children. This review is an overview of what is known from enterovirus and parechovirus infection in children and contains information about the epidemiology, pathogenesis, clinical presentation, diagnosis, treatment, and prognosis of enterovirus and parechovirus infection in children. CONCLUSIONS: EV and HPeV infections are a frequent cause of infection in childhood. The clinical presentation is diverse. RT-qPCR is the best way to detect an EV or HPeV. Cerebrospinal fluid, blood and feces have the highest sensitivity for detecting an EV or HPeV. There is no treatment for EV and HPeV infections. Two vaccines against EV 71 are just licensed in China and will be available on the private market. Little is known about the prognosis of EV and HPeV infections. WHAT IS KNOWN: •EV and HPeV are a frequent cause of infection in children. What is new: •This review gives a brief overview over EV and HPeV infection in children.

OXY-2-15, a novel variant showing increased ceftazidime hydrolytic activity.

OBJECTIVES: Klebsiella oxytoca is a member of the family of Enterobacteriaceae and often contains the ß-lactamase blaOXY gene. Although this ß-lactamase does not naturally hydrolyse ceftazidime, this study describes possible in vivo selection of a clinical K. oxytoca isolate showing increased MICs of ceftazidime. METHODS: To reveal the molecular mechanism underlying this unusual resistance phenotype, WGS, cloning, overexpression, MIC and steady-state kinetic studies were performed. RESULTS: A patient was treated for a septic episode with ceftazidime (4 g/day). This therapy was based on earlier culture results in which, amongst others, a K. oxytoca (Velp-1) isolate was identified. After 11 days of treatment, K. oxytoca Velp-2 was isolated from a pus sample drained from the wound. The isolate showed increased resistance to ceftazidime (MIC ≥64 mg/L) compared with the original K. oxytoca isolate (Velp-1). WGS revealed the presence of a novel blaOXY-2 allele, designated blaOXY-2-15, with a two amino acid deletion at Ambler positions 168 and 169 compared with OXY-2-2. Cloning blaOXY-2-15 into Escherichia coli TOP10 resulted in increased MICs of ceftazidime, but reduced MICs of most other ß-lactams compared with OXY-2-2. Steady-state kinetics confirmed the results of the MIC data, showing clearly significant ceftazidime hydrolysis. CONCLUSIONS: This report shows the risk of in vivo selection of ceftazidime-resistant K. oxytoca isolates after prolonged ceftazidime treatment. Furthermore, it is the first known report of a K. oxytoca isolate conferring resistance to ceftazidime by a two amino acid deletion in the omega loop of OXY-2-2.

Evaluation of the Xpert vanA/vanB assay using enriched inoculated broths for direct detection of vanB vancomycin-resistant Enterococci.

Rapid and accurate detection of VRE (vancomycin-resistant enterococci) is required for adequate antimicrobial treatment and infection prevention measures. Previous studies using PCR for the detection of VRE, including Cepheid's Xpert vanA/vanB assay, reported accurate detection of vanA VRE; however, many false-positive results were found for vanB VRE. This is mainly due to nonenterococcal vanB genes, which can be found in the gut flora. Our goal was to optimize the rapid and accurate detection of vanB VRE and to improve the positive predictive value (PPV) by limiting false-positive results. We evaluated the use of the Xpert vanA/vanB assay on rectal swabs and on enriched inoculated broths for the detection of vanB VRE. By adjusting the cycle threshold (CT) cutoff value to ≤ 25 for positivity by PCR on enriched broths, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 96.9%, 100%, 100%, and 99.5% for vanB VRE, respectively. As shown in this study, CT values of ≤ 25 acquired from enriched broths can be considered true positive. For broths with CT values between 25 and 30, we recommend confirming the results by culture. CT values of >30 appeared to be true negative. In conclusion, this study shows that the Cepheid's Xpert vanA/vanB assay performed on enriched inoculated broths with an adjusted cutoff CT value is a useful and rapid tool for the detection of vanB VRE.

Norovirus disease associated with excess mortality and use of statins: a retrospective cohort study of an outbreak following a pilgrimage to Lourdes.

Although norovirus infection is generally known to be a mild disease, there is some evidence for severe outcome. An outbreak in a Dutch psychiatric institution, originating from pilgrims returning from Lourdes (France), provided an opportunity for performing a retrospective cohort study in order to identify risk factors for norovirus disease and excess mortality. Relative risks (RR) including 95% confidence intervals (CI) showed that attending the pilgrimage (RR 2·0, 95% CI 1·4-3·0) and age >70 (RR 1·7, 95% CI 1·2-2·2) were risk factors for symptomatic infection. In a subset of patients, for whom more detailed information was available, the use of statins was associated with norovirus disease when adjusted for underlying condition (adjusted odds ratio 3·9, 95% CI 1·2-13·0). Mortality was higher in cases infected during the pilgrimage compared to other residents (RR 20·9, 95% CI 4·7-93·8). Norovirus disease can lead to severe outcome. The newly identified risk of statins for contracting norovirus disease may have considerable consequences for the Western world and needs prospective confirmation.

Whole-genome sequencing analysis reveals the spread of a vanB-carrying transposon among different vancomycin-resistant Enterococcus faecium clinical isolates in a non-endemic setting.

BACKGROUND: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements. AIM: To report prospective analysis of whole-genome sequencing (WGS) data, including both core-genome multi-locus sequence typing (cgMLST) and transposon analysis, during a vanB VREfm outbreak. METHODS: Screening for vanB-positive VREfm isolates was performed by real-time polymerase chain reaction (PCR) on an overnight enriched broth and, if positive, subculture was performed. vanB-positive VREfm isolates underwent WGS. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics Workbench, or de-novo assemblies were used for BLASTN comparisons. RESULTS: In total, 1358 real-time PCRs were performed. Two hundred and fifty-one specimens from 207 patients tested positive on PCR for vanB, of which 13 specimens obtained from six patients were identified as vanB VREfm positive on culture. These six patients harboured seven unique isolates belonging to four cluster types: CT118 (N=2), CT2483 (N=3), CT2500 (N=1) and CT2501 (N=1). Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all six patients that could be linked based on epidemiological data. CONCLUSION: A vanB VREfm outbreak occurred in the study hospital, including six patients with isolates belonging to four cluster types. In-depth transposon analysis revealed that dissemination of transposon Tn1549 rather than clonal spread was the cause of the outbreak.

Disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, with remarkable variability in disease severity. Factors determining severity of disease in previously healthy infants are still unclear. It was hypothesized that disease severity is correlated with viral load in primary RSV infection. Infants of a healthy birth cohort were included at signs of their first respiratory tract infection. Nasopharyngeal aspirate was obtained within 48-96 hr and disease severity was assessed with a previously published severity scoring model. PCR was applied to test the aspirates in a semi-quantitative way for the presence of 10 respiratory pathogens. In case of multiple infection, the pathogen with the highest load was defined as the primary pathogen. The correlation between disease severity and viral load was analyzed. A total of 82 infants were included over a period of 2 years. Median age at first respiratory tract infection was 3 months. Pathogens were detected in 77 (94%) infants; more than one pathogen was detected in 35 (43%) infants. RSV was present in aspirates of 30 infants; in 16 aspirates RSV was the primary pathogen. A negative correlation between RSV CT-value and disease severity was found in all RSV cases (rho = -0.52, P = 0.003) and in cases with RSV as the primary pathogen (rho = -0.54, P = 0.03). In conclusion, this is the first report on viral loads in previously healthy infants with RSV infection in the community. Disease severity correlated positively with viral load during primary RSV infection.

Toilet drain water as a potential source of hospital room-to-room transmission of carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.

Practical issues in implementing whole-genome-sequencing in routine diagnostic microbiology.

BACKGROUND: Next generation sequencing (NGS) is increasingly being used in clinical microbiology. Like every new technology adopted in microbiology, the integration of NGS into clinical and routine workflows must be carefully managed. AIM: To review the practical aspects of implementing bacterial whole genome sequencing (WGS) in routine diagnostic laboratories. SOURCES: Review of the literature and expert opinion. CONTENT: In this review, we discuss when and how to integrate whole genome sequencing (WGS) in the routine workflow of the clinical laboratory. In addition, as the microbiology laboratories have to adhere to various national and international regulations and criteria for their accreditation, we deliberate on quality control issues for using WGS in microbiology, including the importance of proficiency testing. Furthermore, the current and future place of this technology in the diagnostic hierarchy of microbiology is described as well as the necessity of maintaining backwards compatibility with already established methods. Finally, we speculate on the question of whether WGS can entirely replace routine microbiology in the future and the tension between the fact that most sequencers are designed to process multiple samples in parallel whereas for optimal diagnosis a one-by-one processing of the samples is preferred. Special reference is made to the cost and turnaround time of WGS in diagnostic laboratories. IMPLICATIONS: Further development is required to improve the workflow for WGS, in particular to shorten the turnaround time, reduce costs, and streamline downstream data analyses. Only when these processes reach maturity will reliance on WGS for routine patient management and infection control management become feasible, enabling the transformation of clinical microbiology into a genome-based and personalized diagnostic field.

[Polymicrobial infections: ecosystems with special properties]. / Polymicrobiële infecties: ecosystemen met bijzondere eigenschappen.

Bacteria often live together in complex communities. Insight into these microbial ecosystems is essential to make it possible to intervene when these ecosystems lead to disease. Bacteria do not only respond to their host, but they also affect each other, which may have far-reaching consequences for the course of the disease. In this article we describe that clinical isolates in a polymicrobial infection can be seen as ecosystems. These ecosystems often have properties that separate isolates do not have; they may, for example, be more virulent or more resistant to antibiotics. We therefore emphasise that the whole is greater than the sum of its parts, even for infections.

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145.

Conserved Citrullinating Exoenzymes in Porphyromonas Species.

Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.

Fosfomycin Etest for Enterobacteriaceae: Interobserver and interlaboratory agreement.

OBJECTIVES: The increasing use of fosfomycin requires reliable susceptibility testing in clinical practice. The reference standard, agar dilution (AD), is rarely used in routine settings. The fosfomycin Etest (BioMérieux) is frequently used, although reading MICs can be hampered by the interpretation of the growth of macrocolonies in the inhibition zone. We investigated the interobserver (IO), interlaboratory (IL), and interobserver-interlaboratory (IOIL) agreement of the fosfomycin Etest and evaluated the agreement with AD. METHODS: Etests were performed for 57 extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae of four bacterial species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae) in two laboratories. Photographs of fosfomycin Etests were interpreted by four observers following manufacturer's instructions. RESULTS: Essential agreement (EA) and categorical agreement (CA) between Etest and AD were 57% and 89% (κ-value 0.68), respectively, with an underestimation of Etest interpretations compared with AD of 0.26 (95% confidence interval [CI] 0.03-0.48) 2-fold dilutions. Between Etest observations, IO-EA and -CA were reached in 82% and 94% of comparisons; IL-EA and -CA in 38% and 85% of comparisons; and IOIL-EA and -CA in 40% and 85% of comparisons, respectively. Agreement of the Etest with AD and between Etests was better for E. coli than for other species. Ignoring all macrocolonies and haze during Etest interpretation improved the agreement with AD (CA κ-value 0.80) and between Etests (CA κ-value from 0.68 to 0.81). CONCLUSIONS: In this study on 57 ESBL-producing Enterobacteriaceae, IOIL agreement was low with an EA of 40% and a CA of 85%, affected most by IL agreement and to a lesser extent by IO agreement.