Caroline Elisabeth Visser

OBJECTIVES: To determine (1) the rate of positive cultures in presumed aseptic nonunions, (2) the rate and microbial spectrum of positive cultures that represented occult infection, and (3) rates of nonunion healing. METHODS: Design: Retrospective cohort study. Setting: Tertiary referral center. Patients Selection Criteria: Adult patients with a presumed aseptic nonunion treated with single-stage revision between 2002 and 2022. Outcome Measures and Comparisons: The rate of positive cultures compared for 2 protocols: old: 1–2 samples cultured 7 days versus new: 5 samples cultured 14 days. The rate of positive cultures meeting occult infection criteria with the new protocol (≥2 samples with phenotypically indistinguishable microorganisms, or ≥1 sample with a high virulent microorganism). Nonunion healing rates between protocols and between groups based on culture results with the new protocol. RESULTS: One hundred seventy-nine patients were included. The rate of positive cultures was 14% (n = 15/105) with the old protocol and 51% (n = 38/74) with the new protocol (P < 0.001). With the new protocol, the rate of positive cultures meeting occult infection criteria was 19% (n = 14/74), and coagulase-negative staphylococci (48%) and Cutibacterium acnes (38%) were the most common microorganisms. Nonunion healing rates after the primary revision did not differ between protocols (old: 82% vs. new: 86%, P = 0.41) and groups based on culture result (sterile: 86% vs. occultly infected: 93%, P = 0.66). The final overall nonunion healing rate was 97%. CONCLUSIONS: Occult infections were identified in 1 in 5 presumed aseptic nonunions using a standardized protocol with 5 intraoperative samples cultured 14 days and were predominantly caused by slow growing, gram-positive microorganisms. The local spectrum and antimicrobial sensitivity of occult infections should be considered when developing empiric antimicrobial protocols. Patients with presumed aseptic nonunions can expect high healing rates, regardless of the culture result. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Early detection of bacterial transmission and outbreaks in hospitals is important because nosocomial infections can result in health complications and longer hospitalization. Current practice to detect outbreaks uses genotyping methods amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS), which are not suitable methods for real-time transmission screening of both susceptible and resistant bacteria. The aim was to assess the typing technique Fourier transform infrared (FTIR) spectroscopy as real-time screening method to discriminate large amounts of susceptible and resistant bacteria at strain level when there is no evident outbreak in comparison with the WGS reference. Isolates of past hospital outbreak strains of Acinetobacter baumannii/calcoaceticus complex (n = 25), Escherichia coli (n = 31), Enterococcus faecium (n = 22), Staphylococcus aureus (n = 37) and Pseudomonas aeruginosa (n = 30) were used for validation of FTIR. Subsequently, Enterococcus faecalis (n = 106) and Enterococcus faecium (n = 104) isolates from weekly routine screening samples when no potential outbreak was present were analysed. FTIR showed reproducibility and congruence of cluster composition with WGS for A. baumannii/calcoaceticus complex and E. faecium outbreak isolates. The FTIR results of E. faecalis and E. faecium isolates from routine samples showed reproducibility, but the congruence of cluster composition with WGS was low. For A. baumannii/calcoaceticus complex and E. faecium outbreak isolates, FTIR appears to be a discriminatory typing tool. However, our study shows the discriminatory power is too low to screen real-time for transmission of E. faecium and E. faecalis at patient wards based on isolates acquired in routine surveillance cultures when there is no clear suspicion of an ongoing outbreak.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics.

Background: Bacteria produce volatile organic compounds (VOCs) during growth, which can be detected by colorimetric sensor arrays (CSAs). The SpecifAST^® system (Specific Diagnostics) employs this technique to enable antibiotic susceptibility testing (AST) directly from blood cultures without prior subculture of isolates. The aim of this study was to compare the SpecifAST^® AST results and analysis time to the VITEK^®2 (bioMérieux) system. Methods: In a 12-month single site prospective study, remnants of clinical positive monomicrobial blood cultures were combined with a series of antibiotic concentrations. Volatile emission was monitored at 37 °C via CSAs. Minimal Inhibitory Concentrations (MICs) of seven antimicrobial agents for Enterobacterales, Staphylococcus, and Enterococcus spp. were compared to VITEK^®2 AST results. MICs were interpreted according to EUCAST clinical breakpoints. Performance was assessed by calculating agreement and discrepancy rates. Results: In total, 96 positive blood cultures containing Enterobacterales, Staphylococcus, and Enterococcus spp. were tested (269 bug–drug combinations). The categorical agreement of the SpecifAST^® system compared to the VITEK^®2 system was 100% and 91% for Gram-negatives and Gram-positives, respectively. Errors among Gram-positives were from coagulase-negative staphylococci. Overall results were available in 3.1 h (±0.9 h) after growth detection without the need for subculture steps. Conclusion: The AST results based on VOC detection are promising and warrant further evaluation in studies with a larger sample of bacterial species and antimicrobials.

Staphylococcus pseudintermedius is an important pathogen in dogs that occasionally causes infections in humans as an opportunistic pathogen of elderly and immunocompromised people. This study compared the genomic relatedness and antimicrobial resistance genes using genome-wide association study (GWAS) to examine host association of canine and human S. pseudintermedius isolates. Canine (n = 25) and human (n = 32) methicillin-susceptible S. pseudintermedius (MSSP) isolates showed a high level of genetic diversity with an overrepresentation of clonal complex CC241 in human isolates. This clonal complex was associated with carriage of a plasmid containing a bacteriocin with cytotoxic properties, a CRISPR-cas domain and a pRE25-like mobile element containing five antimicrobial resistance genes. Multi-drug resistance (MDR) was predicted in 13 (41%) of human isolates and 14 (56%) of canine isolates. CC241 represented 54% of predicted MDR isolates from humans and 21% of predicted MDR canine isolates. While it had previously been suggested that certain host-specific genes were present the current GWAS analysis did not identify any genes that were significantly associated with human or canine isolates. In conclusion, this is the first genomic study showing that MSSP is genetically diverse in both hosts and that multidrug resistance is important in dog and human-associated S. pseudintermedius isolates.

Objectives: Antimicrobial Stewardship Programs commonly have an in-hospital focus. Little is known about the quality of antimicrobial use in hospital outpatient clinics. We investigated the extent and appropriateness of antimicrobial prescriptions in the outpatient clinics of three hospitals. Methods: From June 2018 to January 2019, we performed ten point prevalence surveys in outpatient clinics of one university hospital and two large teaching hospitals. All prophylactic and therapeutic prescriptions were retrieved from the electronic medical records. Appropriateness was defined as being in accordance with guidelines. Furthermore, we investigated the extent to which the dose was adjusted to renal function and documentation of an antibiotic plan in the case notes. Results: We retrieved 720 prescriptions for antimicrobial drugs, of which 173 prescriptions (24%) were prophylactic. A guideline was present for 95% of prescriptions, of which the guideline non-adherence rate was 25.6% (n = 42/164) for prophylaxis and 43.1% (n = 224/520) for therapy. Of all inappropriate prescriptions (n = 266), inappropriate prescriptions for skin and soft tissue infections (n = 60/226) and amoxicillin-clavulanic acid (n = 67/266) made up the largest proportion. In only 13 of 138 patients with impaired or unknown renal function the dosage regimen was adjusted. Amoxicillin-clavulanic acid was the drug for which most often renal function was not taken into account. In 94.6% of prescriptions the antibiotic plan was documented. Conclusions: In hospital outpatient clinics, a substantial part of therapeutics were inappropriately prescribed. Amoxicillin-clavulanic acid was the most inappropriately prescribed drug, due to non-adherence to the guidelines and because dose adjustment to renal function was often not considered.

Background: Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods: Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results: Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion: AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

The prophylactic use of antimicrobial agents to prevent infections in non-surgical situations has hardly been investigated. We investigate the extent, indications and appropriateness of antimicrobial prophylaxis given outside the operating room in a tertiary care hospital. Four point-prevalence surveys were conducted in which all inpatients on that day were screened for the use of prophylactic antimicrobials: medical prophylaxis, prophylaxis around non-surgical interventions and surgical prophylaxis given on the ward. The primary endpoint was the extent of prophylaxis relative to the total number of antimicrobial prescriptions. We also investigated per prescription the presence of a (local) protocol and adherence to these protocols. We registered in total 1020 antimicrobial prescriptions, of which 317 (31.1%) were given as prophylaxis. 827/1020 were antibiotic prescriptions. Of these antibiotic prescriptions, 17.0% was medical prophylaxis, 2.7% prophylaxis around non-surgical interventions and 6.9% surgical prophylaxis administered on a ward. For medical antibiotic prophylaxis, a protocol was present in 125 of 141 prescriptions (88.7%); the protocol was followed in 118 cases (94.4%). For prophylaxis around non-surgical interventions and surgical prophylaxis on the wards, protocol presence and adherence rates were 59.1% and 92.3%, and 73.3% and 97.6% respectively. Of the 96 antiviral and 97 antifungal prescriptions, 42.7% and 57.8%, respectively, were medical prophylaxis, of which 95.1 and 96.3% were prescribed according to protocols respectively. Antimicrobial prophylaxis outside the operating theatre is responsible for a considerable part of total in-hospital antimicrobial use. For most prescriptions there was a protocol and adherence to the protocols was high. The main targets for improvement were prophylaxis around non-surgical interventions and surgical prophylaxis given on the ward.