Whole-genome sequencing enabling the detection of a colistin-resistant hypermutating Citrobacter werkmanii strain harbouring a novel metallo-ß-lactamase VIM-48.

Citrobacter spp. harbouring metallo-ß-lactamases (MBLs) have been reported from various countries and different sources, but their isolation from clinical specimens remains a rare event in Europe. MBL-harbouring Enterobacteriaceae are considered a major threat in infection control as therapeutic options are often limited to colistin. In this study, whole-genome sequencing was applied to characterise five clinical isolates of multidrug-resistant Citrobacter werkmanii obtained from rectal swabs. Four strains possessed a class 1 integron with a novel blaVIM-48 MBL resistance gene and the aminoglycoside acetyltransferase gene aacA4, whilst one isolate harboured a blaIMP-8 MBL. Resistance to colistin evolved in one strain isolated from a patient who had received colistin orally for 8 days. Genomic comparison of this strain with a colistin-susceptible pre-treatment isolate from the same patient revealed 66 single nucleotide polymorphisms (SNPs) and 26 indels, indicating the presence of a mutator phenotype. This was confirmed by the finding of a SNP in the mutL gene that led to a significantly truncated protein. Additionally, an amino acid change from glycine to serine at position 53 was observed in PmrA. Mutations in the pmrA gene have been previously described as mediating colistin resistance in different bacterial species and are the most likely reason for the susceptibility change observed. To the best of our knowledge, this is the first description of a colistin-resistant Citrobacter spp. isolated from a human sample. This study demonstrates the power of applying next-generation sequencing in a hospital setting to trace and understand evolving resistance at the level of individual patients.

Emergence of Citrobacter freundii carrying IMP-8 metallo-ß-lactamase in Germany.

Metallo-ß-lactamases (MBLs) in Enterobacteriaceae are an increasing problem worldwide. This report describes the isolation of Citrobacter freundii carrying IMP-8 MBL from three patients during the period from March 2012 until March 2013 in Germany. The bla IMP-8 enzyme is predominantly found in Asia, where IMP-8 has spread to various enterobacterial species causing serious infections. To our best knowledge, this is the first report of bla IMP-8 habouring Enterobacteriaceae in Europe.

Evaluation of phenotypic detection methods for metallo-ß-lactamases (MBLs) in clinical isolates of Pseudomonas aeruginosa.

Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests.

Acute musculoskeletal infection: comparison of different methods for intraoperative bacterial identification.

PURPOSE OF THE STUDY: Various techniques are used for detection of pathogens in musculoskeletal infection. These methods differ with respect to reliability and ease of handling. A prospective study was performed to evaluate the efficacy of three intraoperative techniques. MATERIAL AND METHODS: In 20 cases (18 patients) with clinically confirmed acute musculoskeletal infections, intraoperative collected swab samples, tissue samples and fluid samples injected into standard blood culture vials were used for microbiological diagnosis. Identification of bacteria, time necessary for detection and ease of handling during surgery was evaluated. RESULTS: In 19 cases bacterial growth was demonstrated using either intraoperative swabs or blood culture technique (95% sensitivity), whereas 18 tissue biopsies were positive (90% sensitivity. 27 bacterial species were isolated. In 18 instances for the swab technique, 14 instances for the tissue biopsy and 4 operations for the blood culture vials, ease of handling was rated as excellent. DISCUSSION: The study demonstrated differences between the three tested methods with respect to ease of handling. With respect to the number of detected organisms and time for their detection there are no significant differences. These last findings are in contrast to of the results of other authors. The reason for this could be that during operative dissection an accurate and specific collection of specimens from the acute deep infected soft tissues and bones independent from the type of surgical procedure is possible. Therefore, even with the swab method a high amount of microorganisms can be recovered. Especially for intraarticular infections, fluid samples injected into standard blood vials is a practical method for the surgeon. In acute musculoskeletal infections other than joint infections, there is less benefit for the blood culture vials. CONCLUSION: Intraoperative swab technique yields valid results comparable to other techniques and is an accurate technique for detection of pathogens from acute musculoskeletal infections. Key words: implant, infections, bacteriological techniques, comparative study.

Metallo-beta-lactamase expressing multi-resistant Acinetobacter baumannii transmitted in the operation area.

Outbreaks of Acinetobacter baumannii demonstrating multiple antibiotic resistance, including meropenem resistance, have been described as severe therapeutic problems. Here we describe a monoclonal outbreak of infection and colonization with multidrug-resistant A. baumannii over a two-month period. Resistance to meropenem was mediated by expression of a metallo-beta-lactamase enzyme. Four of 14 patients showed clinical signs of infection and two died. Contamination of the environment, water, or instruments were excluded as causes of the outbreak. All patients, except one, underwent surgery in a specific operation theatre where surgery of contamination class IV (infected, dirty) was performed. Although individual surgeon error was eliminated, analyses of the patients' histories suggested that bacterial transmission had occurred during surgery. Five patients showed signs of A. baumannii infection and two of these patients suffered from large abdominal wounds infected with a high density of A. baumannii requiring repeated revisions. Presumably, these revisions favoured the transmission of A. baumannii, which is remarkably resistant to various environmental stresses including soaps, disinfectants and dry conditions. No case of meropenem-resistant A. baumannii had been observed in the hospital before the outbreak. Interestingly, the resistant bacteria appear to have been imported by a patient returning from West Africa. This indicates that, similar to MRSA, multiresistant A. baumannii may be introduced by patients from foreign hospitals. The outbreak was stopped in the following months by reinforcing standard procedures and by taking all necessary precautions such as patient isolation, and finally only one new case was detected.