Background Antimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking. Aim We aimed to estimate the occurrence of resistant MG in Belgium. Methods Between July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher’s exact test and logistic regression analysis. Results Of the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics. Conclusion Although limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

•Streptococcus pneumoniae rarely infects the genital tract.•S. pneumoniae as a single bacterial etiology of pelvic inflammatory disease.•In Belgium, since 2006 only 3 serotype 3 S. pneumoniae were reported.

Objectives Antimicrobial resistance of Mycoplasma genitalium (MG) is a growing concern worldwide. Because reliable data on the burden of resistant MG in Belgium are missing, an additional prospective surveillance program was implemented in 2022 to estimate the real burden of resistant MG in Belgium.Methods Belgian laboratories (n=21) provided frozen remnants of MG positive samples to the National Reference Centre of Sexually Transmitted Infections from July to November 2022. The presence of macrolide and fluoroquinolones resistance associated mutations (RAMs) was assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioral variables via Fisher’s exact test and logistic regression analysis.Results Sequencing for both macrolide and fluoroquinolone RAMs was successful for 232/244 MG positive samples. Over half of the samples were resistant to macrolides (55.2%). All MG in samples from men who have sex with men (MSM) (24/24) were resistant to macrolides. The presence of fluoroquinolone RAMs was estimated to be 26% and did not differ with socio-demographic and sexual behaviour characteristics.Conclusions Given the considerable cost of macrolide resistance testing, our data suggest that the use of macrolide resistance testing in MSM does not seem justified in Belgium. However, the lower prevalence of macrolide resistance in other population groups, combined with further emergence of fluoroquinolone resistance provides evidence for macrolide resistance testing in these groups. Continued surveillance of resistance in MG in all groups will be crucial to guide national testing- and treatment strategies.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe prospective study was financed by the Belgian government.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The prospective study has been approved by the Institutional review board of the Institute of Tropical Medicine (ITM) (ref: 1594/22) and the Ethics Committee of the University of Antwerp (ref: 3534).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesThe data supporting the findings of this publication are retained at the Institute of Tropical Medicine, Antwerp and will not be made openly accessible due to ethical and privacy concerns. According to the ITM research data sharing policy, only fully anonymized data can be shared publicly. The data are de-identified (using a unique patient code) but not fully anonymized and it is not possible to fully anonymize them due to the longitudinal nature of the data. Data can however be made available after approval of a motivated and written request to the ITM at ITMresearchdataaccess{at}itg.be. The ITM data access committee will verify if the dataset is suitable for obtaining the study objective and assure that confidentiality and ethical requirements are in place.

Background: Knowledge on the burden attributed to influenza viruses vs other respiratory viruses in children hospitalised with severe acute respiratory infections (SARI) in Belgium is limited. Aim: This observational study aimed at describing the epidemi-ology and assessing risk factors for severe disease. Methods: We retrospectively analysed data from rou-tine national sentinel SARI surveillance in Belgium. Respiratory specimens collected during winter sea-sons 2011 to 2020 were tested by multiplex real-time quantitative PCR (RT-qPCR) for influenza and other respiratory viruses. Demographic data and risk factors were collected through questionnaires. Patients were followed-up for complications or death during hospi-tal stay. Analysis focused on children younger than 15 years. Binomial logistic regression was used to iden-tify risk factors for severe disease in relation to infec-tion status. Results: During the winter seasons 2011 to 2020, 2,944 specimens met the study case definition. Complications were more common in children with underlying risk factors, especially asthma (adjusted risk ratio (aRR): 1.87; 95% confidence interval (CI): 1.46-2.30) and chronic respiratory disease (aRR: 1.88; 95% CI: 1.44-2.32), regardless of infection status and age. Children infected with non-influenza respiratory viruses had a 32% higher risk of complications (aRR: 1.32; 95% CI: 1.06-1.66) compared with children with influenza only. Conclusion: Multi-virus testing in chil-dren with SARI allows a more accurate assessment of the risk of complications and attribution of burden to respiratory viruses beyond influenza. Children with asthma and respiratory disease should be prioritised for clinical care, regardless of their virological test result and age, and targeted for prevention campaigns.

BackgroundTo support the COVID-19 pandemic response, many countries, including Belgium, implemented baseline genomic surveillance (BGS) programs aiming to early detect and characterize new SARS-CoV-2 variants. In parallel, Belgium maintained a sentinel network of six hospitals that samples patients with severe acute respiratory infections (SARI) and integrated SARS-CoV-2 detection within a broader range of respiratory pathogens. We evaluate the ability of the SARI surveillance to monitor general trends and early signals of viral genetic evolution of SARS-CoV-2 and compare it with the BGS as a reference model.MethodsNine-hundred twenty-five SARS-CoV-2 positive samples from patients fulfilling the Belgian SARI definition between January 2020 and December 2022 were sequenced using the ARTIC Network amplicon tiling approach on a MinION platform. Weekly variant of concern (VOC) proportions and types were compared to those that were circulating between 2021 and 2022, using 96,251 sequences of the BGS.ResultsSARI surveillance allowed timely detection of the Omicron (BA.1, BA.2, BA.4, and BA.5) and Delta (B.1.617.2) VOCs, with no to 2 weeks delay according to the start of their epidemic growth in the Belgian population. First detection of VOCs B.1.351 and P.1 took longer, but these remained minor in Belgium. Omicron BA.3 was never detected in SARI surveillance. Timeliness could not be evaluated for B.1.1.7, being already major at the start of the study period.ConclusionsGenomic surveillance of SARS-CoV-2 using SARI sentinel surveillance has proven to accurately reflect VOCs detected in the population and provides a cost-effective solution for long-term genomic monitoring of circulating respiratory viruses.

Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods’ reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5–100

Objectives: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. Methods: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. Results: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. Conclusion: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

Background: Knowledge on the burden attributed to influenza viruses versus other respiratory viruses in children hospitalized with severe acute respiratory infections (SARI) in Belgium is limited. This observational study aimed at describing the epidemiology and assessing risk factors for severe disease.Methods: We retrospectively analyzed data from routine national sentinel SARI surveillance in Belgium. Respiratory specimens collected during the 2011-2020 winter seasons were tested by multiplex RT-qPCRs for influenza and other respiratory viruses. Patient demographics and risk factors were collected through questionnaires. Patients were followed-up for the occurrence of complications or death during hospital stay. The study case definition included patients with acute respiratory illness with onset within the last ten days, measured or reported fever, cough and/or dyspnoea, and requiring hospitalization at least overnight. Analysis focused on children <15 years of age. Binomial logistic regression was used to identify risk factors for severe disease in relation to infection status.Findings: During the 2011-2020 winter seasons, 2,944 specimens met the study case definition. Complications were more common in children with underlying risk factors, especially asthma (adj. risk ratio 1.87 (1.46, 2.30)) and chronic respiratory disease (adjusted risk ratio 1.88 (1.44, 2.32)), regardless of infection status and age. Children infected with non-influenza respiratory viruses had a 32% higher risk for complications (adjusted risk ratio 1.32 (1.06, 1.66)) compared to children with influenza only.Interpretation: Multi-virus testing in children with SARI allows a more accurate assessment of the risk for complications and attribution of burden to respiratory viruses beyond influenza. Children with asthma and respiratory disease should be prioritized for clinical care regardless of their virological test result and age, and targeted for prevention campaigns.Trial Registration Details: The protocol was registered on ClinicalTrials.gov with identifier NCT05620953.Funding Information: This study was supported by Belgian Federal Public Service ‘Health, Food Chain Safety, and Environment’, Belgian National Insurance Health Care (INAMI/RIZIV), Regional Health Authorities (Flanders: AZG, Brussels: COCOM, Wallonia: AVIQ).Declaration of Interests: The authors declare no conflict of interest.Ethics Approval Statement: The study surveillance protocol was approved by a central Ethical Committee (reference 2012/310; in 2011: Centre Hospitalier Universitaire St-Pierre, Brussels, Belgium; since 2014: Universitair Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium) and the local ethical committees of each participating hospital each year. Informed consent was obtained from all participants or parents/guardians.

BACKGROUND:Seasonal human coronaviruses (hCoVs) broadly circulate in humans. Their epidemiology and effect on the spread of emerging coronaviruses has been neglected thus far. We aimed to elucidate the epidemiology and burden of disease of seasonal hCoVs OC43, NL63, and 229E in patients in primary care and hospitals in Belgium between 2015 and 2020. METHODS:We retrospectively analysed data from the national influenza surveillance networks in Belgium during the winter seasons of 2015-20. Respiratory specimens were collected through the severe acute respiratory infection (SARI) and the influenza-like illness networks from patients with acute respiratory illness with onset within the previous 10 days, with measured or reported fever of 38°C or greater, cough, or dyspnoea; and for patients admitted to hospital for at least one night. Potential risk factors were recorded and patients who were admitted to hospital were followed up for the occurrence of complications or death for the length of their hospital stay. All samples were analysed by multiplex quantitative RT-PCRs for respiratory viruses, including seasonal hCoVs OC43, NL63, and 229E. We estimated the prevalence and incidence of seasonal hCoV infection, with or without co-infection with other respiratory viruses. We evaluated the association between co-infections and potential risk factors with complications or death in patients admitted to hospital with seasonal hCoV infections by age group. Samples received from week 8, 2020, were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). FINDINGS:2573 primary care and 6494 hospital samples were included in the study. 161 (6·3%) of 2573 patients in primary care and 371 (5·7%) of 6494 patients admitted to hospital were infected with a seasonal hCoV. OC43 was the seasonal hCoV with the highest prevalence across age groups and highest incidence in children admitted to hospital who were younger than 5 years (incidence 9·0 [95% CI 7·2-11·2] per 100 000 person-months) and adults older than 65 years (2·6 [2·1-3·2] per 100 000 person-months). Among 262 patients admitted to hospital with seasonal hCoV infection and with complete information on potential risk factors, 66 (73·3%) of 90 patients who had complications or died also had at least one potential risk factor (p=0·0064). Complications in children younger than 5 years were associated with co-infection (24 [36·4%] of 66; p=0·017), and in teenagers and adults (≥15 years), more complications arose in patients with a single hCoV infection (49 [45·0%] of 109; p=0·0097). In early 2020, the Belgian SARI surveillance detected the first SARS-CoV-2-positive sample concomitantly with the first confirmed COVID-19 case with no travel history to China. INTERPRETATION:The main burden of severe seasonal hCoV infection lies with children younger than 5 years with co-infections and adults aged 65 years and older with pre-existing comorbidities. These age and patient groups should be targeted for enhanced observation when in medical care and in possible future vaccination strategies, and co-infections in children younger than 5 years should be considered during diagnosis and treatment. Our findings support the use of national influenza surveillance systems for seasonal hCoV monitoring and early detection, and monitoring of emerging coronaviruses such as SARS-CoV-2. FUNDING:Belgian Federal Public Service Health, Food Chain Safety, and Environment; Belgian National Insurance Health Care (Institut national d'assurance maladie-invalidité/Rijksinstituut voor ziekte-en invaliditeitsverzekering); and Regional Health Authorities (Flanders Agentschap zorg en gezondheid, Brussels Commission communautaire commune, Wallonia Agence pour une vie de qualité).

BackgroundSeasonal influenza-like illness (ILI) affects millions of people yearly. Severe acute respiratory infections (SARI), mainly influenza, are a leading cause of hospitalisation and mortality. Increasing evidence indicates that non-influenza respiratory viruses (NIRV) also contribute to the burden of SARI. In Belgium, SARI surveillance by a network of sentinel hospitals has been ongoing since 2011.AimWe report the results of using in-house multiplex qPCR for the detection of a flexible panel of viruses in respiratory ILI and SARI samples and the estimated incidence rates of SARI associated with each virus.MethodsWe defined ILI as an illness with onset of fever and cough or dyspnoea. SARI was defined as an illness requiring hospitalisation with onset of fever and cough or dyspnoea within the previous 10 days. Samples were collected in four winter seasons and tested by multiplex qPCR for influenza virus and NIRV. Using catchment population estimates, we calculated incidence rates of SARI associated with each virus.ResultsOne third of the SARI cases were positive for NIRV, reaching 49.4% among children younger than 15 years. In children younger than 5 years, incidence rates of NIRV-associated SARI were twice that of influenza (103.5 vs 57.6/100,000 person-months); co-infections with several NIRV, respiratory syncytial viruses, human metapneumoviruses and picornaviruses contributed most (33.1, 13.6, 15.8 and 18.2/100,000 person-months, respectively).ConclusionEarly testing for NIRV could be beneficial to clinical management of SARI patients, especially in children younger than 5 years, for whom the burden of NIRV-associated disease exceeds that of influenza.

Background: Zoonotic coronaviruses have repeatedly taken the spotlight leading to severe global epidemics over the last two decades. In addition, seasonal coronaviruses (sCoVs) broadly circulate in humans. Their epidemiology could have broad impacts on the spread of emerging coronaviruses, but has been neglected so far.Methods: Clinical samples and data were collected from hospitalized patients with severe acute respiratory infection (SARI) and primary care patients with influenza-like illness (ILI), recruited through the national influenza surveillance networks in Belgium. Multiplex RT-qPCRs for respiratory viruses, including sCoVs OC43, NL63 and 229E, and SARS-CoV-2 were performed. Incidence rates of sCoV infection between 2015-2020 were estimated by season and age group. The impact of co-infections and comorbidities on the outcome of hospitalized patients was assessed.Findings: Hospitalized children under five carry the highest burden of disease for OC43 (IR =9·0, 95%CI 7·2-11·2 per 100,000 person-months) and NL63 (IR=5·2, 95%CI 3·9-6·9 per 100,000 person-months), while adults over 65 carry the highest burden of disease for 229E (IR=1·7, 95%CI 1·3-2·2 per 100,000 person-months). In hospitalized children under five, complications were associated with co-infections (p=0·02). Overall, comorbidities were strongly associated with a severe outcome following sCoV infection (p=0·006). In early March 2020, the SARI surveillance detected the first SARS-CoV-2-positive sample concomitantly with the first confirmed COVID-19 case without travel history to China. The ILI surveillance system captured two peaks in the number of primary care visits at weeks five (influenza) and 12 (SARS-CoV-2).Interpretation: We show that sCoVs can cause severe complications and death, especially in combination with pre-existing comorbidities and/or co-infections. Furthermore, we encourage the leverage of national influenza surveillance systems for early detection and monitoring of emerging coronaviruses such as SARS-CoV-2.Funding: Federal Public Service 'Health, Food Chain Safety, and Environment', National Insurance Health Care (INAMI/RIZIV), Regional Health Authorities (Flanders: AZG, Brussels: COCOM, Wallonia: AVIQ).Declaration of Interests: The authors declare no conflict of interest.Ethics Approval Statement: The study surveillance protocol was approved by a central Ethical Committee (reference AK/12-02- 11/4111; in 2011: Centre Hospitalier Universitaire St-Pierre, Brussels, Belgium; since 2014: Universitair Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium) and the local ethical committees of each participating hospital. Informed consent was obtained from all participants or parents/guardians.

INTRODUCTION:Clostridium species is a group of anaerobic bacteria constituting the colonic microflora of the intestinal tract. Since molecular methodologies based on 16 rRNA have been established for the classification and the recognition of bacterial species, more than 150 species of Clostridium have been described. Most are considered harmless saprophytes; however, these bacteria may be involved in a wide variety of infections and may be a common cause of enteritis and enterotoxemias in humans.CASE PRESENTATION:We present the case of a 60-year-old Asian patient admitted in the emergency room with an acute appendicitis where a blood culture showed the presence of a Clostridium hathewayi. This microorganism is an anaerobic bacteria described in 2001 as a Gram negative end-pointed bacillus, usually endospore-forming. It was reclassified in 2014 as Hungatella hathewayi. A literature review has been performed to find articles relating to this bacteria in a clinical case.CONCLUSION:C. hathewayi is microorganism recently reclassified as Hungatella hathewayi. Its growth in blood cultures has been reported in a few cases in the literature. Although only a few articles have reported its involvement in clinical infections, we assess that its part in the cause of the illness should be evaluated.

IMP-13 is a class B metallo-β-lactamase that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa from Italy.1 The IMP-13 gene was mostly described as part of the class 1 InPSG integron located on a Tn5051 transposon, and it was reported in intercontinentally disseminating P. aeruginosa of sequence type 621 causing outbreaks in Italy, Romania and Argentina.1–3 Multidrug-resistant P. aeruginosa are at the origin of outbreaks that are often difficult to control and to eradicate. The persistence of pools of transferable elements carrying antimicrobial resistance genes in hidden environmental reservoirs may further explain the resurgence of some epidemics, as well as their ability to quickly appear. The role of Pseudomonas putida as a metallo-β-lactamase reservoir was recently reinforced by showing its ability to transfer carbapenem resistance to P. aeruginosa.4 We briefly describe here the carriage of blaIMP-13 in Pseudomonas monteilii, a species closely related to P. putida, recovered from the clinical environment.

Sir, IMP-13 is a class B metallo-b-lactamase that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa from Italy. The IMP-13 gene was mostly described as part of the class 1 InPSG integron located on a Tn5051 transposon, and it was reported in intercontinentally disseminating P. aeruginosa of sequence type 621 causing outbreaks in Italy, Romania and Argentina. – 3 Multidrug-resistant P. aeruginosa are at the origin of outbreaks that are often difficult to control and to eradicate. The persistence of pools of transferable elements carrying antimicrobial resistance genes in hidden environmental reservoirs may further explain the resurgence of some epidemics, as well as their ability to quickly appear. The role of Pseudomonas putida as a metallo-b-lactamase reservoir was recently reinforced by showing its ability to transfer carbapenem resistance to P. aeruginosa. We briefly describe here the carriage of blaIMP-13 in Pseudomonas monteilii, a species closely related to P. putida, recovered from the clinical environment. In the setting of an outbreak due to a GES-12-producing Acinetobacter baumannii occurring in 2009, the inanimate environment of an acute care burn unit of a Belgian hospital was regularly screened. In January 2011, the materials and surfaces in multiple hospital environments were sampled. In total, .70 different specimens were obtained from both dry inanimate surfaces (floor, bed frame, bed linen and computer pads) and wet surroundings (sinks, baths, toilets, toilet brushes and soap). This screening was carried out all over the burn unit as well as in the operating rooms, corridors and nurse restrooms. Screening cultures were grown overnight at 358C on a MacConkey plate onto which a 30 mg ceftazidime disc (Bio-Rad, Nazareth, Belgium) was placed. While no A. baumannii could be recovered from any of the specimens, culture of a toilet brush sample yielded the growth of a ceftazidime-resistant isolate that was presumptively identified as P. putida by Vitek 2 GN-ID cards. However, this isolate was subsequently confirmed as P. monteilii both by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (Microflex LT, Bruker Daltonics, Leipzig, Germany) and by sequencing of the gene encoding 16S rRNA (100% identity with P. monteilii GAPP1, accession no. GU396289), emphasizing the difficulty of correctly distinguishing between P. putida and P. monteilii by conventional biochemical methods. Antimicrobial susceptibility testing by agar disc diffusion according to CLSI guidelines revealed a multidrugand carbapenem-resistant isolate, which only remained susceptible to colistin and intermediately susceptible to aztreonam. MICs, determined according to the CLSI microdilution reference method, confirmed the highlevel resistance of this environmental isolate to extendedspectrum b-lactamases (ESBLs; MICs of ceftazidime and cefepime .128 mg/L, and MICs of imipenem and meropenem .32 mg/L). Double discs showed the presence of a synergy between imipenem (10 mg) and EDTA (465 mg; 10 mL of 0.125 M EDTA stock), which highly suggested the presence of a metallo-b-lactamase. PCR sequencing confirmed the presence of a blaIMP-13 gene located upstream of a mutated gene encoding an aminoglycoside-modifying enzyme, aacA4-2, embedded in a new class I integron, In449, which is slightly different (six mutations in 1889 nucleotides analysed; accession number JN091097) from the InPSG already described in P. aeruginosa. Moreover, PCR performed with primers specific to the transposase (tnpA) and the resolvase (tnpR) of Tn5051 were negative, indicating that this integron is located on a different and unknown genetic structure. Neither genes encoding ESBLs (blaSHV, BEL, VEB, PER and GES) nor oxacillinase of groups 1, 2 or 10 could be detected by PCR. Plasmid extractions performed using the Kieser method or with Qiagen midi preparation did not yield any plasmidic bands and several attempts to obtain IMP-13-expressing electroporants in PA01 wild-type P. aeruginosa failed, suggesting the possible location of blaIMP-13 on the bacterial chromosome. Subsequent to this screening, reinforced infection control measures were taken, including discarding of all toilet brushes, reviewing and extensive explanation to the housekeeping personnel of the standard operation cleaning/disinfection procedures, implementation of antibacterial filters on taps, reinforced practice of hand hygiene disinfection, daily cleaning of the entire unit, systematic environmental screening of infected/colonized patients’ rooms upon discharge, enhanced

During a PCR-based surveillance study of β-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumannii isolates were detected at 6 Belgian hospitals. DNA sequencing of the bla(GES) genes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n = 2), Egypt (n = 2), and Palestinian territories (Gaza) (n = 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located bla(GES-11) and bla(GES-12) and a likely chromosomally located novel variant bla(GES-14)) were detected as part of a class 1 integron, also including the aac6'Ib and dfrA7 genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing bla(GES-11) and bla(GES-12). Cloning of the bla(GES) genes in Escherichia coli identified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various bla(GES-like) genes, especially those conferring carbapenem resistance in A. baumannii and its importation in Western Europe from Middle Eastern countries.

Carbapenems are among the drugs of choice for treating nosocomial infections due to multidrug-resistant Acinetobacter baumannii strains (4). However, their efficacy is being increasingly compromised by the spread of carbapenem-resistant isolates, mostly following acquisition of carbapenemases of class D (OXA-23, OXA-40-like, and OXA-58 oxacillinases) (1, 6, 8, 9). Outbreaks of OXA-23-producing A. baumannii isolates are increasingly being reported worldwide (7, 8). In these isolates, blaOXA-23 is associated with an upstream-inserted insertion sequence, ISAba1, which is responsible for its mobilization and expression. In two unrelated A. baumannii isolates from France, the blaOXA-23 gene was associated with another insertion sequence, ISAba4 (3). Here, we describe the first ISAba4-blaOXA-23-expressing A. baumannii isolates in Belgium. In July 2007, a 69-year-old Belgian resident was admitted to the intensive care unit (ICU) of a general hospital in Morocco with a diagnosis of pneumonia and cardiac failure. The patient was transferred to the ICU of Saint-Joseph Hospital (Gilly, Belgium), and culture of respiratory secretions grew a carbapenem-resistant and multidrug-resistant A. baumannii strain, GIL1. Despite prompt treatment with colistin, the patient died. The A. baumannii isolate was identified with the API 32GN system (BioMerieux, Marcy-l'Etoile, France) and confirmed by detection of the intrinsic blaoxa-51/69-like gene (10). According to MIC determination by Etest (AB Biodisk, Solna, Sweden), A. baumannii GIL1 was resistant to all β-lactams (2), including imipenem and meropenem (MIC, >32 μg/ml), sulbactam (MIC, 48 μg/ml), ciprofloxacin (MIC, >32 μg/ml) and amikacin (MIC, >256 μg/ml); borderline susceptible to tigecycline (MIC, 6 μg/ml) and rifampin (MIC 3 μg/ml); and fully susceptible to colistin only (MIC, 0.5 μg/ml). PCR analysis followed by sequencing revealed that A. baumannii GIL1 harbored the blaOXA-23 gene, along with the naturally and chromosomally located blaOXA-69 gene and a novel blaADC allele (5), blaADC-39 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU652244,term_id:186969836,term_text:EU652244}}EU652244). blaADC-39 differed from the A. baumannii AYE blaAmpC gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CT025798,term_id:88758819,term_text:CT025798}}CT025798) by only two nucleotide substitutions (A1022C and G1135A) leading to two amino acid changes (N351T and D379N). Isoelectric focusing confirmed the expression of OXA-23 (pI 6.3 to 6.5) and ADC-39 β-lactamase (pI >8.5) (not shown). Using a previously described PCR approach (3), ISAba1 was found inserted upstream of blaADC-39 gene but not upstream of the blaOXA-23 or blaOXA-69 gene. Instead, another insertion sequence, ISAba4, was found upstream of the blaOXA-23 gene, with a genetic organization similar to that described for Tn2007: i.e., orf3 and mobA genes surrounding the ISAba4-blaOXA-23-ATPase fragment inserted in orf1 (3) (Fig. ​(Fig.11). FIG. 1. Genetic organization of the three β-lactamase genes found in A. baumannii GIL1. (A) ISAba4-blaOXA-23. (B) ISAba1-blaADC-39. (C) blaOXA-69. Horizontal arrows represent open reading frames and their transcriptional orientations. Promoters brought ... The blaOXA-23 gene was encoded on a 25-kb plasmid that could be electroporated into rifampin-resistant A. baumannii CIP7010 but was not self-transferable by conjugation. Only the blaOXA-23 gene was transferred in the electroporants, as revealed by susceptibility testing. Since the initial detection of this strain, six other patients of the ICU have been identified as carriers or infected with ISAba4-blaOXA-23-expressing A. baumannii isolates. These isolates were indistinguishable by pulsed-field gel electrophoresis, suggesting clonal diffusion. Furthermore, pulsed-field gel electrophoresis analysis revealed that A. baumannii GIL1 presented identical banding patterns to one ISAba4-blaOXA-23 A. baumannii isolate reported in France, thus illustrating its propensity to disseminate and underscoring the need for epidemic surveys geared to estimate the true prevalence of these ISAba4-OXA-23 producers. ISAba1 and ISAba4 have been suggested to be at the origin of blaOXA-23 gene mobilization and to provide strong promoter sequences for their expression (3). Whether ISAba4 may procure a fitness advantage over ISAba1 in A. baumannii strains in terms of transposition frequency or level of expression needs to be further investigated.