Evaluation of phenotypic tests for detection of Amp C beta-lactamases in clinical isolates from a tertiary care hospital of Rawalpindi, Pakistan.

OBJECTIVE: To evaluate sensitivity and specificity of disc approximation test compared to three-dimensional extract test as a phenotypic gold standard test for detection of AmpC beta-lactamase producing Escherichia coli and Klebsiella pneumoniae. METHODS: The cross-sectional validation study was conducted from November 2014 to April 2015 at Army Medical College, Rawalpindi, Pakistan. Extended spectrum beta lactamases (ESBLs) were isolated from various clinical specimens. Screening for AmpC beta-lactamases was done by using cefoxitin disc (30µg) showing inhibition zone diameter of <18mm. Screen-positive isolates were subjected to disc approximation test (DAT) and three-dimensional extract test(3-DET).SPSS 20 was used for statistical analysis. RESULTS: A total of 120 ESBL producing Gram negative rods were included in the study. Out of these 120, 82(68.33%) were found to be AmpC beta-lactamase producing on screening with cefoxitin disc. Amongst these 82 isolates, Escherichia coli were identified in 57(69.51%) and Klebsiella pneumoniae in 25 (30.48%). Phenotypic confirmation by disc approximation test (DAT) identified 43(52.43%). AmpC beta-lactamase producing isolates, whereas gold standard 3-DET showed 38(46.34%) of AmpC beta-lactamase producing isolates. Hence, sensitivity of disc approximation test (DAT) was found to be 88%, specificity was 92%, positive predictive value was 92.68%, negative predictive value was 87.80% and diagnostic accuracy was 90.24%. CONCLUSIONS: Implementation of disc approximation test in the laboratories can help in identifying AmpC beta-lactamase harbouring organisms.

Methicillin resistant coagulase negative staphylococcus: From colonizer to a pathogen.

The objective of our study was to determine the frequency of methicillin resistance in coagulase negative Staphylococcus (CoNS) and to determine its in-vitro antimicrobial susceptibility to various other routinely used antibiotics. It was a cross sectional study conducted at the department of Microbiology, Army Medical College, Rawalpindi, Pakistan from June 2011 to May 2012. The organisms were identified on the basis of colony morphology, Gram staining, catalase, DNAase and slide/tube coagulase tests. The organisms were considered to be methicillin resistant when the diameter of zone of inhibition was less than 25mm around 30µg cefoxitin disc. Antibiotic sensitivity was determined using the Modified Kirby-Bauer disc diffusion method. From a total of 337 CoNS, 201 were methicillin resistant and were included in the study. All were resistant to Penicillin, followed by Erythromycin (93•1%), Ciprofloxacin (77%), Co-trimoxazole (74•8%), Gentamicin (68•3%), Clindamycin (51•06%), Tetracycline (44•6%), Fusidic acid (40%), Rifampicin (39•5%), Chloramphenicol (19•3%), Linezolid (2%), Minocycline (1•1%), and Vancomycin (0%). More than half of CoNS were methicillin resistant. Vancomycin is the only drug to which all of the MRCoNS were sensitive, with more than 98% of the isolates being sensitive to Linezolid and Minocycline.

Coagulase negative staphylococci - a fast emerging threat.

OBJECTIVE: To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. METHODS: The descriptive cross-sectional study was carried out at Army Medical College, Rawalpindi, from June 2009 to May 2012, and comprised clinical samples mostly from patients admitted to the intensive care unit. They were inoculated onto appropriate culture media depending upon the specimen. After 24-hour incubation at 35°C, coagulase-negative staphylococci were identified on the basis of colony morphology, gram staining, a positive catalase and a negative tube coagulase test.Methicillin resistance among the isolated staphylococci was determined using a 30µg Cefoxitin disc as per the Clinical and Laboratory Standards Institute protocol. Number of coagulase-negative staphylococci for each year and their methicillin resistance rates were calculated. A comparison was made with methicillin resistant staphylococcus aureus) isolated during the same period. RESULTS: Of the total 1331 specimens studies over three years, 581(43.65%) were coagulase-negative staphylococci. The rate of coagulase-negative staphylococci and methicillin resistance was higher each year; 110(26.6%) in May 2009-Jun 2010, 134(36.5%) in 2011, and 337(61%) in 2012. Methicillin resistance rates also increased from 25(22.7%) to 46(34.3%) and then to 201(59.6%) in 2012.Maximum isolated specimens came from blood 311(53.5%), followed by pus/swabs 204(35.1%). CONCLUSIONS: The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise.

Efficacy of Antimicrobials Against Multidrug-Resistant Acinetobacter baumannii from Patients in a Tertiary Care Hospital.

Introduction: In just two decades Acinetobacter baumannii has attained considerable importance, evolving from an insignificant organism to a leading pathogen especially in intensive care unit settings globally. Treatment options are already very limited and have almost run out due to the rapid emergence of antimicrobial resistance. Evaluation of antimicrobials that are currently in use to determine their effectiveness against multidrug-resistant (MDR) strains and developing newer options is of utmost importance. We thus set out to determine the efficacy of routinely used antibiotics against MDR A. baumannii. Materials and Methods: This was a cross-sectional study conducted at the Department of Microbiology, Army Medical College, National University of Medical Sciences (Rawalpindi, Pakistan) from December 2015 to June 2016. The organisms were identified on the basis of colony morphology, gram staining, catalase, oxidase, motility test, and API (analytical profile index) 20NE. The organisms were considered to be MDR when the isolate was found to be resistant to at least one agent in more than three antimicrobial groups. Antibiotic sensitivity was determined using the modified Kirby-Bauer disc diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines. Results: The 77 isolates were found to have good sensitivity to tigecycline (94.8%) and minocycline (80.5%). Most of the isolates were resistant to other routinely used antibiotics. Conclusion: A few antibiotics, tigecycline and minocycline, are still effective against these MDR A. baumannii. We need to remain up to date regarding the efficacy of antibiotics to effectively treat patients with these MDR bacteria.

Comparative Efficacy of Ceftaroline with Linezolid against Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus.

OBJECTIVE: To compare the in vitro antimicrobial efficacy of ceftaroline with linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus. STUDY DESIGN: Quasi-experimental study. PLACE AND DURATION OF STUDY: Microbiology Department, Army Medical College, Rawalpindi, from January to December 2013. METHODOLOGY: Clinical samples from respiratory tract, blood, pus and various catheter tips routinely received in the Department of Microbiology, Army Medical College, Rawalpindi were innoculated on blood and MacConkey agar. Staphylococcus aureus was identified by colony morphology, Gram reaction, catalase test and coagulase test. Methicillin resistant Staphylococcus aureus detection was done by modified Kirby Bauer disc diffusion method using cefoxitin disc (30 µg) and the isolates were considered methicillin resistant if the zone of inhibition around cefoxitin disc was ≤ 21 mm. Bacterial suspensions of 56 Staphylococcus aureus isolates and 50 MRSA isolates were prepared, which were standardized equal to 0.5 McFarland's turbidity standard and inoculated on Mueller-Hinton agar plates followed by application of ceftaroline and linezolid disc (Oxoid, UK), according to manufacturer's instructions. The plates were then incubated at 37 °C aerobically for 18 - 24 hours. Diameters of inhibition zone were measured and interpretated as per Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Out of 106 isolates all of the 56 Staphylococcus aureus (100%) were sensitive to ceftaroline and linezolid. However, out of 50 methicillin resistant Staphylococcus aureus, 48 (96%) were sensitive to ceftaroline whereas, 49 (98%) were sensitive to linezolid. CONCLUSION: Ceftaroline is equally effective as linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus.

In Vitro Efficacy of Doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii by E-Test.

OBJECTIVE: To assess the in vitro efficacy of doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii using Epsilometer strips. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi and National University of Sciences and Technology, Islamabad, from May 2014 to September 2014. METHODOLOGY: A total of 60 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa collected from various clinical samples received from Military Hospital were included in the study. The specimens were inoculated onto blood, MacConkey and chocolate agars. The isolates were identified using Gram staining, motility, catalase test, oxidase test and API 20NE (Biomeriux, France). Organisms identified as Acinetobacter baumannii and Pseudomonas aeruginosa were included in the study. Bacterial suspensions equivalent to 0.5 McFarland turbidity standard of the isolates were prepared and applied on Mueller Hinton agar. Epsilometer strip was placed in the center of the plate and incubated for 18-24 hours. Minimum Inhibitory Concentration (MIC) was taken to be the point where the epsilon intersected the E-strip. MIC of all the isolates was noted. RESULTS: For Pseudomonas aeruginosa isolates, MIC(50) was 12 µg/mL and MIC(90) was 32 µg/mL. For Acinetobacter baumannii MIC(50) and MIC(90) was 32 µg/mL. CONCLUSION: Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting.