FALSE POSITIVE PHENOTYPIC DETECTION OF METALLO-BETA-LACTAMASES IN ACINETOBACTER BAUMANNII.

Phenotypic detection of metallo-ß-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.

[Carbapenemases of gram-negative bacteria]. / Karbapenemaze gram-negativnih bakterija.

Carbapenems are often antibiotics of last resort for the treatment of severe infections. They are stable to most beta-lactamases produced by gram-negative bacteria. However, bacterial enzymes named carbapenemases can efficiently hydrolyze carbapenems. They are produced most frequently by Enterobacteriaceae and non-fermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannnii. They belong to group A (KPC, SME, IMI, NMC), B (VIM, IMP, SPM, GIM, NDM, SIM, DIM, AIM) and D (OXA-23, OXA-24, OXA-48, OXA-58, OXA-143). The accurate and rapid laboratory identification of carbapenem-resistant isolates is important to prevent spread of such multidrug resistant strains and to avoid therapeutic failures. Therapeutic options are often limited because carbapenemases are encoded on mobile genetic elements which often harbour resistance genes to other groups of antibiotics. Thus, colistin is often the only therapeutic option.

Emergence of colistin resistance in Enterobacter aerogenes from Croatia.

A colistin-resistant Enterobacter aerogenes [study code 12264] was isolated from the tracheal aspirate of a 71-year-old male patient in the General Hospital [GH] in Pula, Croatia. The patient was previously treated in University Hospital Centre in Rijeka with colistin in order to eradicate Acinetobacter baumannii isolate, susceptible only to colistin and tigecycline. Genes encoding ESBLs [blaTEM, blaSHV, blaCTX-M, blaPER-1] were screened by PCR. The strain was shown to possess blaCTX-M-15 and blaTEM-1 genes. To asses genes possibly involved in resistance to colistin the chromosomal enconding mgrB gene and the plasmid-mediated mcr-1 and mcr-2 genes were screened as described previously. Mcr-1 and mcr-2 genes were not detected and mgrB gene presented a wild-type sequence. PCR-based Replicon typing method [PBRT] conducted on an E. aerogenes isolate, showed that the strain carried an IncN plasmid. Adaptive mechanisms such as changes of the bacterial cell outer membrane that cause porin decrease or presence of an efflux pump, due to selection pressure exerted by the therapeutic administration of colistin, could be responsible for the development of colistin resistance in our strain, as recently reported in E. aerogenes from France. Due to effective infection control measures, the colistin-resistant strain did not spread to other patients or hospital wards. This is the first report of an ESBL-producing, colistin-resistant E. aerogenes in clinically relevant samples such as endotracheal aspirate and blood culture, showing the presence of this rare resistance profile among Gram-negative bacteria.

Emergence of different Acinetobacter baumannii clones in a Croatian hospital and correlation with antibiotic susceptibility.

OBJECTIVES: During routine diagnostic laboratory work, the clinical microbiologist observed an increase of Acinetobacter baumannii isolates with three different carbapenem susceptibility patterns: susceptible, intermediate and resistant. Isolates belonging to the same carbapenem susceptibility phenotype exhibited identical susceptibility/resistance patterns to non-ß-lactam antibiotics. This prompted us to analyse the mechanisms of carbapenem-resistance and the molecular epidemiology of the isolates. A total of 59 A. baumannii isolates were analysed and grouped according to their susceptibility to imipenem: group 1 were susceptible (N=24), group 2 were intermediate (N=8) and group 3 were resistant (N=27) to imipenem. MATERIAL AND METHODS: PCR and sequencing was used to detect resistance genes. Genotyping of the isolates was performed by PFGE and MLST. RESULTS: Out of 27 resistant isolates, 20 harboured blaOXA-40-like and 7 blaOXA-23-like genes. ISAba1 was found upstream of blaOXA-51 and blaOXA-23 genes. PFGE genotyping demonstrated the existence of three major A. baumannii clones in GH Pula and determination of sequence groups showed that the isolates belonged to international clones commonly associated with multidrug-resistance. MLST (performed on six isolates) showed diverse population structure of isolates belonging to the same cluster, including ST 195, ST 231, ST 775 and ST 1095. CONCLUSIONS: A previous study conducted in 2009-2010 showed that reduced susceptibility to carbapenems in GH Pula was only associated with upregulation of the intrinsic OXA-51 ß-lactamase. In this study a shift to isolates with acquired oxacillinases, belonging to two major clones was reported.

[Subjective general assessment of nutritional status in patients with chronic renal failure and regular hemodialysis]. / Subjektivna opca procjena nutritivnog statusa u bolesnika s kronicnim zatajenjem bubrega na redovnoj hemodijalizi.

PATIENTS AND METHODS: The protein energy malnutrition rating has been investigated in 75 hemodialysis (HD) patients with average treatment period of 67.3 month. A method of subjective global assessment (SGA) of nutritional status was used including body weight and food intake changes evaluated in last six months, as well as the nutritional status in relation to the subcutaneous fat loss and muscle mass wasting. SGA classifies the patients as: A well-nourished, B--mildly malnourished and C--severely malnourished. RESULTS: Malnutrition was identified in 40%, mild in 34.7% and severe in 5.3% of the patients. In the group of patients with normal nutritional status there were significantly fewer patients with lower values of objective parameters of nutritional status (body mass index (BMI), mid-arm muscle circumference (MAMC), phase angle from bioimpedance measurement, serum albumin) than in the group of patients with severe malnutrition. The differences in these parameters were of marginal significance between the groups with normal nutritional status and with mild malnutrition. The former had a significantly higher BMI value and serum albumin concentrations in comparison to the malnourished patients. There was also a considerable difference in the mean value of C-reactive protein (CRP) among all groups. The rates of malnutrition defined by the SGA method showed a high negative correlation with BMI, MAMC and serum albumin, and high positive correlation with CRP. CONCLUSIONS: We confirmed the SGA method to be simple to use, and to correlate strongly with other parameters of nutrition. We suggest that the CRP test be included in the assessment of nutritional status to determine the patient inflammatory status, considering the etiologic association between inflammation and malnutrition.