Investigation of Carbapenemases in Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae Strains Isolated in 2014 in Turkey

Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confirmed by combination disk synergy tests. Types of carbapenemases were investigated using specific primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available

Detection of a New Resistance-Mediating Plasmid Chimera in a bla(OXA-48)-Positive Klebsiella pneumoniae Strain at a German University Hospital

Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven bla(OXA-48) gene-carrying Klebsiella pneumoniae isolates, which were isolated between 2013 and 2014 at the University Medical Center in Gottingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively; and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All bla(OXA-48) genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in K. pneumoniae in Germany

The changing nature of aminoglycoside resistance mechanisms and prevalence of newly recognized resistance mechanisms in Turkey

Objective, To determine the most frequently. occurring individual and combined resistance mechanisms, in Gram-negative bacteria resistant to any of the clinically available aminoglycosides in Turkey, and to compare these mechanisms with those found in smaller, earlier studies. Methods Aminoglycoside resistance mechanisms in Gram-negative isolates, resistant to either gentamicin, tobramycin, netilmicin or amikacin collected in different regions of Turkey were evaluated both phenotypically and genotypically using 12 aminoglycosides, and up to 22 aminoglycoside resistance gene probes. Results Among 696 aminoglycoside-resistant Gram-negative bacteria, resistance rates were very high for gentamicin (94.5%), tobramycin (82.4%). netilmicin (53.6%), and amikacin (49.7%). Although isepamicin was the most active aminoglycoside, against Gram-negative bacteria, increased resistance (29.7%) was found and resistance rates were higher than those in most of the other countries surveyed in earlier studies. The most common aminoglycoside resistance mechanisms (AAC(3)-II (GTN), AAC(6)-I (TNA), and ANT(2)-I (GT)) in the earlier studies were also found in the present isolates of Klebsiella spp., Enterobacter spp. and Escherichia coli, with increased complexity. In addition to these old mechanisms, two new aminoglycoside resistance mechanisms, namely AAC(6)-III (TNAI) and AAC(6)-IV (GTNA), were also found at significant frequencies (11.9% and 26.9%, respectively) in these isolates of Enterobacteriaceae (n = 435). Among the isolates of Pseudomonas spp. (n = 150), in addition to the increased complexity of enzymatic resistance mechanisms (AAC(3)-I (16.6%), AAC(6')-II (29.3%), AAC(6)-III (19.3%), ANT(2)-I (40%)), permeability resistance seemed to be responsible for the high rates of resistance to, aminoglycosides. Conclusion The results of this. study indicated increased resistance to clinically available aminoglycosides, including isepamicin, even though it was, the most active, as a result of both the presence of new aminoglycoside resistance mechanisms and the increased, complexity of all mechanisms, including permeability resistance, particularly in Pseudomonas in Turkey

Simple and reliable detection of slime production of Candida spp. directly from blood culture bottles: Comparison of visual tube method and transmission electron microscopy

Early detection of slime production may be useful for clinical decision because of its suggestive property for potential pathogenic capacity of a Candida strain especially in patients with a prosthetic device. In this study we aimed to compare the visual tube method (VTM) with transmission electron microscopy (TEM) in order to confirm the reliability of the former method. In order to demonstrate the reproducibility of the tube method and to determine the correct timing for the test, Candida isolates directly obtained from blood culture (DBC) bottles and their two subsequent subcultures were used. The results of this study showed that VTM is a simple and reliable method which can be used in every clinical mycology laboratory, provided that the test is applied on DBC isolates; as the ability of slime production is decreased or lost even after the first subculturing. We suggest that this simple method can be used and may have some contributions to the ongoing studies on the controversial issue concerning removal of biomaterials in candidemic patients

Evaluation of two commercial methods for rapid detection of the carbapenemase-producing Klebsiella pneumoniae

In this study, we evaluated the performance of the two commercial methods (Rapidec Carba NP and NG-Test Carba-5) for the rapid detection of carbapenemase-producing Klebsiella pneumoniae. A total of 224 Klebsiella pneumoniae strains previously characterized for carbapenemase genes by polymerase chain reaction were included in the study. The strain collection included 30 non-carbapenemase producers, 85 OXA-48-like, 59 NDM, 14 IMP, 7 KPC, 7 VIM, 19 OXA-48-like plus NDM, and 3 KPC plus NDM producers. Rapidec Carba NP and NG-Test Carba 5 was performed according to the manufacturer's instructions. NG-Test Carba 5 correctly detected all carbapenemase-producing K. pneumoniae, however, Rapidec Carba NP failed to detect 41% of OXA-48-like producers even with extended incubation time. The overall sensitivity and specificity were 81,9% and 100% for Rapidec Carba NP, 100% and 100% for NG-Test Carba 5, respectively. Both tests seem to be fast and reliable for the detection of carbapenemase-producing K. pneumoniae especially for microbiology laboratories where molecular tests cannot be performed. However, Rapidec Carba NP with weak hydrolysis activity for OXA-48 like might be used in regions where OXA-48 is not prevalent. The additional advantage of NG-Test Carba 5 is that it specifically detects carbapenemases giving way to threat-related infections with an effective drug such as ceftazidime-avibactam or meropenem- vaborbactam