Antimicrobial susceptibility profiles of anaerobic bacteria, isolated from human clinical specimens, within different European and surrounding countries. A joint ESGAI study.

OBJECTIVES: Studies on the antimicrobial susceptibility profile of anaerobic bacteria are underrepresented in the literature. Within this study we aim to give an extensive overview of the differences in antimicrobial susceptibility profiles between different European and surrounding countries. METHODS: Minimal inhibitory concentration (MIC) data of different antibiotics were collected from 10 participating laboratories, representing an equal number of countries. All MIC's were determined using Etest, according to the protocol used by the participating laboratory. Anaerobic genera represented by at least 10 clinical isolates were included in the study. RESULTS: Each country tested different antibiotics, sometimes depending on the kind of infection and/or the anaerobic species isolated. All countries tested clindamycin and metronidazole. Resistance rates differed remarkably between the different countries. Especially in Kuwait, resistance was high for all tested antibiotics. Unexpected metronidazole resistance was observed for Finegoldia magna isolates, Peptoniphilus isolates and Eggerthella lenta isolates. CONCLUSIONS: Due to the extensive differences in antimicrobial susceptibility profile of anaerobic bacteria isolated within different countries, we strongly recommend to perform this kind of study on a regular basis.

A cluster of Legionnaires' disease in Belgium linked to a cooling tower, August-September 2016: practical approach and challenges.

A cluster of Legionnaires' disease (LD) with 10 confirmed, three probable and four possible cases occurred in August and September 2016 in Dendermonde, Belgium. The incidence in the district was 7 cases/100 000 population, exceeding the maximum annual incidence in the previous 5 years of 1.5/100 000. Epidemiological, environmental and geographical investigations identified a cooling tower (CT) as the most likely source. The case risk around the tower decreased with increasing distance and was highest within 5 km. Legionella pneumophila serogroup 1, ST48, was identified in a human respiratory sample but could not be matched with the environmental results. Public health authorities imposed measures to control the contamination of the CT and organised follow-up sampling. We identified obstacles encountered during the cluster investigation and formulated recommendations for improved LD cluster management, including faster coordination of teams through the outbreak control team, improved communication about clinical and environmental sample analysis, more detailed documentation of potential exposures obtained through the case questionnaire and earlier use of a geographical information tool to compare potential sources and for hypothesis generation.

In Vitro Susceptibility of Burkholderia cepacia Complex Isolated from Cystic Fibrosis Patients to Ceftazidime-Avibactam and Ceftolozane-Tazobactam.

We tested the in vitro susceptibility of ceftazidime-avibactam and ceftolozane-tazobactam and 13 other antibiotics against 91 Burkholderia cepacia complex (BCC) strains isolated from cystic fibrosis patients since 2012. The highest susceptibility (82%) was found for trimethoprim-sulfamethoxazole. Eighty-one and 63% of all BCC strains were susceptible to ceftazidime-avibactam and ceftolozane-tazobactam, respectively. For temocillin, ceftazidime, piperacillin-tazobactam, and meropenem, at least 50% of the strains were susceptible. B. stabilis seems to be more resistant than other BCC species.

Validation of MALDI-TOF MS Biotyper database optimized for anaerobic bacteria: The ENRIA project.

Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates. Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia, Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical relevance of these less common anaerobic bacteria.

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS.

Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories.

Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates.

Whole genome sequencing (WGS) enables detailed characterization of bacteria at single nucleotide resolution. It provides data about acquired resistance genes and mutations leading to resistance. Although WGS is becoming an essential tool to predict resistance patterns accurately, comparing genotype to phenotype with WGS is still in its infancy. Additional data and validation are needed. In this retrospective study, we analysed 234 E. coli isolates from positive blood cultures using WGS as well as microdilution for 11 clinically relevant antibiotics, to compare the two techniques. We performed whole genome sequencing analyses on 234 blood culture isolates (genotype) to detect acquired antibiotic resistance. Minimal inhibitory concentrations (MIC) for E. coli were performed for amoxicillin, cefepime, cefotaxime, ceftazidime, meropenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, gentamicin, tobramycin, and ciprofloxacin, using the ISO 20776-1 standard broth microdilution method as recommended by EUCAST (phenotype). We then compared the two methods for statistical 'agreement'. A perfect (100%) categorical agreement between genotype and phenotype was observed for gentamicin and meropenem. However, no resistance to meropenem was observed. A high categorical agreement (> 95%) was observed for amoxicillin, cefepime, cefotaxime, ceftazidime, amikacin, and tobramycin. A categorical agreement lower than 95% was observed for amoxicillin/clavulanic acid, piperacillin/tazobactam, and ciprofloxacin. Most discrepancies occurred in isolates with MICs within ± 1 doubling dilution of the breakpoint and 22.73% of the major errors were samples that tested phenotypically susceptible at higher antibiotic exposure and were therefore considered as 'not resistant'. This study shows that WGS can be used as a valuable tool to predict phenotypic resistance against most of the clinically relevant antibiotics used for the treatment of E. coli bloodstream infections.

Sequence-based typing of Legionella pneumophila serogroup 1 clinical isolates from Belgium between 2000 and 2010.

Sequence-based typing (SBT) is a discriminatory method widely used to genotype Legionella pneumophila strains. A total of 86 clinical L. pneumophila serogroup 1 (sg1) isolates, collected between January 2000 and December 2010 in the two Belgian National Reference Centres for Legionella pneumophila, were genotyped using the internationally standardised SBT protocol of the European Working Group for Legionella Infections (EWGLI). The isolates could be classified into 31 different sequence types (ST, index of diversity: 0.879). The obtained STs were submitted to the EWGLI SBT-database for L. pneumophila. In our study, ST47 (27.9%) and ST1 (19.8%) were the most frequently detected STs. The detected profiles were a combination of both frequently isolated and unique STs, and of both worldwide distributed and more local strains. Two STs, ST880 and ST881, were new to the EWGLI database. In conclusion, we characterised L. pneumophila sg1 isolates with the SBT method, and created a Belgian profile database that will be useful for future epidemiological studies.

Molecular characterization of extraintestinal and diarrheagenic Escherichia coli blood isolates.

Pathogenic E. coli strains can be classified into two major groups, based on the presence of specific virulence factors: extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC). Several case reports describe that DEC can cause bloodstream infections in some rare cases. This mainly concerns a few specific sequence types that express virulence factors from both ExPEC and DEC. In this study, we retrospectively analysed 234 E. coli blood isolates with whole genome sequencing (WGS). WGS was performed on an Illumina NovaSeq6000. Genotyping was performed using BioNumerics software. The presence of genes was determined with a minimum percentage sequence identity (ID) threshold of 95% and a minimum length for sequence coverage of 95%. Three of the 234 (1.28%) isolates were defined as DEC, 182 (77.78%) as ExPEC, and 49 (20.94%) did not carry pathotype-associated virulence genes. We identified 112 different virulence genes, 48 O-antigens, and 28 H-antigens 82 STs, among the 234 analyzed isolates. ST131 and ST88 were related to healthcare-associated infections. This study provides insight into the prevalence of virulence factors in a large set of E. coli blood isolates from the UZ Brussel. It illustrates high diversity in virulence profiles and highlights the potential of DEC to carry virulence factors associated with extraintestinal infections, making it possible for unusual pathotypes to invade and survive in the bloodstream causing bacteraemia. Diarrheagenic strains causing bacteremia are rare and presently underreported, but modern sequencing techniques will better underscore their importance.

Sink drains as reservoirs of VIM-2 metallo-ß-lactamase-producing Pseudomonas aeruginosa in a Belgian intensive care unit: relation to patients investigated by whole-genome sequencing.

BACKGROUND: Hospital-acquired infections caused by VIM-encoded metallo-ß-lactamase-positive Pseudomonas aeruginosa are a major problem in intensive care units (ICUs) worldwide. A previous study conducted in the UZ Brussel hospital revealed that sink drains of the ICU were a possible source of various multidrug-resistant pathogenic bacteria. AIM: To investigate the presence and persistence of VIM P. aeruginosa in the sink drains of the four adult ICUs and their role in nosocomial infections, emphasizing sink-to-patient transmission. METHODS: Thirty-six sinks located in the ICUs of the UZ Brussel were sampled and screened for the presence of VIM P. aeruginosa in August and October 2019. Whole-genome sequencing (WGS) was performed on all positive sink drain isolates together with 61 isolates from patients who were retrospectively selected (ICU patients 2019-2020, N = 46; non-ICU patients 2019, N = 6). FINDINGS: Twenty sinks were found positive for P. aeruginosa at both sampling time-points. WGS revealed that the predominating environmental cluster belonged to sequence type ST111. Ten additional STs were identified. VIM-2 was detected among all ST17 (N = 2) and ST111 (N = 14) sink drain isolates. Based on whole-genome multi-locus sequence typing analysis of all genomes, 15 clusters of highly related isolates were identified, of which seven included both sink drain and clinical isolates. CONCLUSION: Our findings confirm that sink drains are a possible source of VIM-2 P. aeruginosa, probably after being contaminated with clinical waste from patients. Patients could be exposed to VIM-2 P. aeruginosa dispersed in their environment because of colonized sink drains.

Serratia marcescens outbreak in a neonatal intensive care unit and the potential of whole-genome sequencing.

BACKGROUND: Serratia marcescens is notorious for its increasing antimicrobial resistance and potential to cause outbreaks in neonatal intensive care units (NICUs). A promising tool in outbreak investigations is whole-genome sequencing (WGS). OBJECTIVES: To describe a S. marcescens outbreak (2018-2019) in an NICU and discuss which infection control measures contributed to containment, addressing the potential of WGS. METHODS: S. marcescens isolates from patients and the environment isolated during the 2018-2019 NICU outbreak were analysed. In comparison, isolates from previous presumed NICU outbreaks and adult blood cultures were included. WGS and whole-genome multi-locus sequence typing analysis were performed. RESULTS: Sixty-three S. marcescens isolates were analysed. The 2018-2019 outbreak was divided into three clusters, including four environmental strains (drains, N=3; baby scale, N=1). The strains differed significantly from those of an NICU outbreak in 2014 and adult blood cultures. Besides standard infection control measures, the siphons were replaced and weekly decontamination was performed with acetic acid 10%. Seven acquired-resistance genes and 29 virulence-associated genes were detected. CONCLUSIONS: It was assumed that both neonates and drains were reservoirs of S. marcescens cross-contamination via the hands of healthcare workers and parents. Initially, standard measures, including hand hygiene, were reinforced. However, definitive containment was achieved only after replacement of the siphons and weekly decontamination with acetic acid. WGS enables faster recognition of an outbreak with accurate mapping of the spread, facilitating the implementation of infection control measures. WGS also provides interesting information about the spread of antibiotic resistance and virulence genes.

An overview of the data obtained during the validation of an optimized MALDI-TOF MS Biotyper database for the identification of anaerobic bacteria.

This data in brief article presents the data obtained during the validation of the optimized Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) database. The validation was performed by the different expertise laboratories, collaborating within the European Network for the Rapid Identification of Anaerobes (ENRIA) project, using 6309 human clinical anaerobic bacterial strains. Different databases were compared with each other; the db 5989 database (V5 database); the V5 database complimented with Main Spectral Profiles (MSPs) of ENRIA strains added to the next update of the database; and the V5 database complimented with the MSPs of all anaerobic clinical isolates collected within the ENRIA project. For a comprehensive discussion of the full dataset, please see the research article that accompanies this data article (Veloo et al., 2018) [1].

Outbreak of multidrug-resistant Acinetobacter baumannii in a Belgian university hospital after transfer of patients from Greece.

Multidrug-resistant (MDR) Acinetobacter baumannii are emerging as important nosocomial pathogens. These organisms have a capacity for long-term survival in the hospital environment. The purpose of this study was to describe the course and control of an outbreak with MDR A. baumannii in a Belgian university hospital after transfer of two trauma patients from Greece. Wounds in both patients were colonised with MDR A. baumannii. Over an 11 month period from September 2004 to July 2005, carbapenem-non-susceptible A. baumannii (producing carbapenem-hydrolysing oxacillinase OXA-58) were isolated from 28 patients, despite early implementation of contact precautions. MDR A. baumannii was detected in routine clinical diagnostic samples from 26 patients and in screening specimens from an additional two patients. Twenty patients (71.4%) were colonised or infected during their stay in intensive care. Twenty-four (85.7%) respiratory samples were positive for MDR A. baumannii. Careful review of all procedures related to the respiratory tract did not identify a common route of transmission. Outbreak control required multiple interventions, including contact isolation of colonised and infected patients, monitoring the practice of personnel, screening of asymptomatic patients, use of isolation rooms and enhanced environmental disinfection. Introduction of single-use ventilator circuits was considered but the outbreak was controlled before implementation.

The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci.

Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.

Cyclosporine nephrotoxicity: comparative cytochemical study of rat kidney and human allograft biopsies.

The aim of this study was to detect early renal changes in the rat. Female Wistar rats received oral doses of cyclosporine (12.5, 25 or 50 mg/kg daily). The duration of the experiment was 1, 2, and 3 weeks. Controls received the vehicle only (olive oil). The following alterations were seen by light microscopy: Hypertrophy of the juxtaglomerular apparatus (PAS stain). Cytoplasmic droplets of neutral fat (Oil Red 0) in clusters of cortical tubules, probably belonging to the same nephron. Both the above phenomena increased with dosage and duration of treatment and were absent in controls. In the fat containing tubulus (FCT) brush border staining (alkaline phosphatase) was decreased or absent. Since after PAS the brush border was visualized in many FCT, it is concluded that many FCT were proximal tubulus (PT) of which the brush border has been damaged. In FCT mitochondrial staining (Cytochrome oxidase activity) was strongly decreased or absent. Mean lysosomal volume (acid phosphatase and dipeptidase II) is increased in the PT; in some cyclosporine animals, lysosomes were enlarged, while in others they were comparable to controls. Electron microscopy showed in some PT cells an increased number of empty vacuoles and focal alteration of mitochondria. Normal mitochondria were present next to grossly altered mitochondria. Autophagocytosis of mitochondria was clearly present. The lysosomes appeared swollen and contained electron dense material, not organised in the typical 50 A pattern of myeloid figures. These morphological changes suggest a defect of mitochondrial metabolism, leading to lipid deposition in PT. The mitochondrial metabolism can be disturbed by a direct toxic effect of cyclosporine or indirectly via ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxigenic Corynebacterium ulcerans in human and non-toxigenic Corynebacterium diphtheriae in cat.

Corynebacterium diphtheriae and Corynebacterium ulcerans are rarely isolated from clinical samples in Belgium. A case of toxigenic C. ulcerans in a woman is described, which confirms that this pathogen is still present. During investigation of the patient's cats, only a non-toxigenic toxin-bearing C. diphtheriae strain was detected.

Salmonella enterica subspecies houtenae serotype 44:z4, z23:--as a rare cause of meningitis.

Reptiles can carry and shed the bacterium Salmonella without showing any signs of illness. Transmission occurs by ingesting Salmonella after handling a reptile or objects contaminated by a reptile. Young children are especially vulnerable to Salmonella infection and can experience serious complications. We describe a case of reptile-associated Salmonella meningitis in a 2.5-month-old infant.

Oligoclonal bands in cerebrospinal fluid detected by PhastSystem isoelectric focusing.

Pharmacia's "PhastSystem" for semi-automated isoelectric focusing (IEF) in thin precast polyacrylamide gels (PAGE) was found to be as sensitive as high-resolution protein electrophoresis (HRPE) in agarose gels and conventional PAGE-IEF for detection of oligoclonal banding (OB) in concentrated cerebrospinal fluid (CSF) samples. Both PhastSystem IEF and HRPE revealed OB in CSF from eight of nine multiple sclerosis patients and four of 10 patients with various types of infection of the central nervous system as opposed to only two of 70 patients with miscellaneous neuropsychiatric disorders. The PhastSystem also frequently detected OB in silver-stained, unconcentrated CSF from patients with multiple sclerosis.

Phytol and peroxisome proliferation.

Infantile Refsum's disease is characterized by high levels of phytanic acid and the absence of normal hepatic peroxisomes. We investigated the in vivo influence of phytol, a precursor of phytanic acid, on peroxisomes by both biochemical and morphological methods. Enhanced supply of phytol in the diet of adult mice causes proliferation of hepatic peroxisomes. The peroxisomal beta-oxidizing capacity as well as exchanges of acyl moieties between peroxisomes and mitochondria are raised around 5- and 2-fold, respectively. In parallel a 1.5-fold increase of total catalase and mitochondrial butyryl-CoA dehydrogenase activities occurs, whereas peroxisomal urate oxidase and glycolate oxidase remain normally active. Serum triglyceride levels are decreased after 3 wk of phytol feeding; serum cholesterol levels remain unaffected. Phytol feeding also induces peroxisome proliferation in duodenal epithelium, in myocardium and in skin sebaceous glands, but not in kidney.