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Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae. Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 μg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro, especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness.

Background Beta-lactamase enzymes-producing Enterobacteriaceae have emerged in many hospital settings resulting in poor treatment outcomes. We aimed to determine resistant patterns of Beta-lactamase enzymes among Enterobacteriaceae collected from referral hospitals in Khartoum state, Sudan. Methods A total of 168 Enterobacteriaceae recovered from clinical samples of patients during May 2014 to February 2015. Identification and susceptibility testing of the isolates were performed as per standard methods. Double-disk synergy test was applied to determine the presence of extended-spectrum β-lactamase (ESBL) production. AmpC beta-lactamases and carbapenemase were screened using AmpC disk test and the modified Hodge test, respectively. Results ESBL-producing Enterobacteriaceae represented 45.2%, with a higher rate among K. pneumoniae. AmpC beta-lactamase detected as 49.3%, with peak levels among Acinetobacter baumannii (A. baumannii) (83.3%) and Enterobacter cloacae (75%). Carbapenemase production was found among 74.5% of isolates, with high rates among A. baumannii (89%) and K. pneumoniae (78%). Overall Enterobacteriaceae, highest resistance rates were found in penicillins and cephalosporins agents. Amikacin and imipenem revealed good activities against most of the isolates, except for A. baumannii (66.7% and 75%, respectively). E. coli yielded high resistance rates for amoxicillin (98.8%), amoxicillin-clavulanic acid (93.8%), cefotaxime (93.8%), and ciprofloxacin (76.5%). Moderate resistance rates were observed among K. pneumoniae for ciprofloxacin (61.5%), nitrofurantoin (57.7%) and cefoxitin (40.4%). Conclusions ESBL, AmpC beta-lactamase and carbapenemase-producing Enterobacteriaceae are emerging and may contribute to increasing antimicrobial resistance in Sudan. Phenotypic screening of such enzymes is rapid and straightforward and should be simultaneously done and carried out routinely in our hospitals.

Staphylococcus aureus remains a leading, virulent pathogen capable of expressing complex drug resistance that requires up to 2-4 days for laboratory analysis. In this study, we evaluate the ability of automated microscopy of immobilized live bacterial cells to differentiate susceptible from non-susceptible responses of S. aureus isolates (MRSA/MSSA, clindamycin resistance/susceptibility and VSSA/hVISA/VISA) to an antibiotic based on the characterization of as few as 10 growing clones after 4 h of growth, compared to overnight growth required for traditional culture based methods. Isolates included 131 characterized CDC isolates, 3 clinical isolates and reference strains. MRSA phenotype testing used 1 h of 1 μg/mL cefoxitin induction followed by 3 h of 6 μg/mL cefoxitin. Clindamycin susceptibility testing used 1h of induction by 0.1 μg/mL erythromycin followed by 3h of 0.5 μg/mL clindamycin. An automated microscopy system acquired time-lapse dark-field images, and then computed growth data for individual immobilized progenitor cells and their progeny clones while exposed to different test conditions. Results were compared to concurrent cefoxitin disk diffusion and D-test references. For CDC organisms, microscopy detected 77/77 MRSA phenotypes and 54/54 MSSA phenotypes, plus 53/56 clindamycin-resistant and 75/75 clindamycin susceptible strains. Automated microscopy was used to characterize heterogeneous and inducible resistance, and perform population analysis profiles. Microscopy-based hVISA population analysis profiles (PAPs) were included as an extended proof of concept, and successfully differentiated VSSA from hVISA and VISA phenotypes compared to plate-based PAP.

Background: Many clinical laboratories have problems detecting various β-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. It is more imperative to use various phenotypic methods for detection of various β-lactamases in routine microbiology laboratory on day-to-day basis to prevent antimicrobial resistance by evidence-based judicious use of antimicrobials. Aims: In view of the multidrug-resistant organisms being reported world over, we planned a cross-sectional prospective analytical study to determine resistance mechanism by various β-lactamases in Gram-negative clinical isolates using various phenotypic methods. Materials and Methods: All nonrepeat, nonenteric clinical isolates of Gram-negative bacilli, resistant to at least two third-generation cephalosporins, were first screened by Novel disc placement method, and isolates showing multiple mechanisms of resistance and reduced zone of inhibition for imipenem were further confirmed for AmpC and metallo β-lactamases. Statistical Analysis: All the data was managed and analyzed in Microsoft Excel. Results: Out of 807 isolates tested, as many as 795 (98.51%) revealed the presence of extended-spectrum β-lactamases (ESBLs). Only 10 isolates of Escherichia coli and 2 of Klebsiella pneumoniae did not show production of ESBL. A total of 450 (55.76%) isolates produced single enzyme,while 345 (42.75%) strains revealed multiple enzyme production simultaneously. Only ESBL production was seen in 315 (39.03%) strains, only AmpC in 75 (9.29%) and only MBL in 60 (7.44%) strains, while ESBL and AmpC together were seen in 219 (27.14%) and AmpC plus MBL in 92 (11.40%) strains. However, ESBL plus MBL were never observed together. All three enzymes were simultaneously detected in 34 (4.21%) strains. Conclusion: This innovative method of disc placement makes it easy, affordable, and reliable method for routine use by basic microbiology laboratories for detection of various β-lactamases, pending confirmation for AmpC and metallo β-lactamase by three-dimensional test and double disc potentiation test, respectively.