The potential of different molecular biology methods in tracking clones of Acinetobacter baumannii in an ICU setting.

PURPOSE: This study aimed to characterize A. baumannii strains isolated from patients in an intensive care unit (ICU) setting. Molecular techniques were used to study clonal relatedness and determine a fast, efficient and cost-effective way of detecting persistent clones. METHODOLOGY: A. baumannii (n=17) were obtained in June and November 2015 from a single ICU setting in South India. DNA typing methods such as multilocus sequence typing (MLST), single-locus sequence-based typing (SBT) and DNA fingerprinting PCRs (M13, DAF4 and ERIC2) were employed to understand the association of clones. PCRs were performed for the antimicrobial resistance genes ISAba1-blaOXA-51-like, ISAba1-blaOXA-23-like, blaNDM-1, blaPER-7 and blaTEM-1, and the virulence genes cpa 1, cpa2 and pkf. RESULTS: The MLST showed some degree of corroboration with the other DNA typing methods. The M13 PCR was found to give better results than the other fingerprinting methods. ST848 (CC92) was the dominant strain isolated in both June and November. All isolates were blaOXA-51-like-positive, with 16 having ISAba1 upstream of the blaOXA-51-like and blaOXA-23-like genes. Genes such as blaNDM-1 (23 %, n=4), blaPER-7 (58.8 %, n=10), pkf (82 %, n=14), blaTEM-1 (5.8 %, n=1), cpa1 (5.8 %, n=1) and cpa2 (5.8 %, n=1) were also detected. CONCLUSION: M13 PCR can be used in routine environmental surveillance for the detection of persistent antibiotic resistant clones in an ICU setting because of its reliability and simplicity. Further studies based on greater sample size, conducted at the multi-centre level, can give us a better understanding of the reliability of the molecular methods that can be used for the detection of persistent clones in the hospital setting.

First report of blaOXA-181-mediated carbapenem resistance in Aeromonas caviae in association with pKP3-A: Threat for rapid dissemination.

OBJECTIVES: Carbapenemase-producing Aeromonas spp. are of great concern in healthcare settings and are also known to acquire clinically relevant resistance genes. In this study, carbapenem-non-susceptible Aeromonas isolates were characterised for their molecular mechanisms of resistance. METHODS: Among 180 Aeromonas isolates, 10 carbapenem-non-susceptible isolates were selected based on their antimicrobial susceptibility profile. Carbapenemase production was investigated by the CarbaNP test. ESBL-, AmpC- and carbapenemase-encoding genes were screened by PCR. Isolates VBF557 and VBF856 with high MICs for imipenem were selected for whole-genome sequencing (WGS). Conjugation experiments were performed to determine the transmissibility of resistance. RESULTS: WGS remarkably revealed the presence of class D ß-lactamases (AmpS/AmpH), class C ß-lactamases and class B2 metallo-ß-lactamase (cphA3) in VBF557. In contrast, VBF856 had multiple resistance genes coding for aminoglycoside, sulphonamide, carbapenem (blaOXA-181 class D ß-lactamase), macrolide, fluoroquinolone, rifampicin, phenicol, tetracycline and trimethoprim resistance. This is the first global report of blaOXA-181 in Aeromonas spp. Interestingly, blaOXA-181 was identified in association with transposon Tn2013 in plasmid pKP3-A. Additionally, an IncQ2 plasmid with qnrS2 was identified. Among the tested isolates, VBF1116 and VBF888 possessed blaNDM and blaVEB, respectively, by PCR. None of the other isolates harboured any tested ß-lactamase genes. The resistance gene was transmissible in the presence of imipenem. CONCLUSIONS: Presence of such resistance genes in plasmids further adds complexity for control of spread of carbapenem resistance. This study reveals the emergence of carbapenem resistance among Aeromonas spp. and the importance of mobile genetic elements such as plasmids in interchanging resistance determinants between species.

Strengths and limitations of various screening methods for carbapenem-resistant Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience.

Carbapenemase-mediated carbapenem resistance is a major concern across the world. Rapid detection of carbapenemase-producing organisms is of great importance in clinical settings. However, it is essential to have a test with good sensitivity and specificity. The aim of the study was to compare the performance of RAPIDEC® CARBA NP and modified carbapenem inactivation method (mCIM) recommended by Clinical and Laboratory Standards Institute guideline 2017. A total of ninety carbapenem resistant Escherichia coli and Klebsiella pneumoniae have been tested. The presence of various carbapenemases was screened by conventional multiplex polymerase chain reaction. RAPIDEC® CARBA NP detected 90%, whereas mCIM detected 99% of the study isolates tested. Although RAPIDEC® CARBA NP is a rapid test, the sensitivity is reduced for blaOxa-48Likedetection; while mCIM could pick up blaOxa-48Likeenzymes with excellent sensitivity. Further, organisms producing low carbapenemase activity enzymes, thickness of the inoculum and the disc potency are likely to influence the test results of mCIM with an overnight delay.

First Report on the Draft Genome Sequences of Corynebacterium diphtheriae Isolates from India.

We report here the draft genome sequences of five Corynebacterium diphtheriae isolates of Indian origin. The C. diphtheriae isolates TH1141, TH510, TH1526, TH1337, and TH2031 belong to sequence type ST-50, ST-295, ST-377, ST-405, and ST-405, with an average genome size of 2.5 Mbp.

A Pilot Study on Carbapenemase Detection: Do We See the Same Level of Agreement as with the CLSI Observations.

INTRODUCTION: Rapid identification of carbapenemase producing organisms is of great importance for timely detection, treatment and implementation of control measures to prevent the spread. The Modified Hodge Test (MHT) and Carba NP test is recommended by CLSI for the detection of carbapenemases in Enterobacteriaceae. However, MHT may give false positive results or fail to detect metallo ß-lactamases (MBLs). In the US, MHT is the most widely used test for detection of carbapenemases and has been found to have a sensitivity and specificity of >90% for bla KPC producers. However, in India, the prevalence of bla NDM is higher than bla KPC producers. AIM: To evaluate the usefulness of CarbaNP in an Indian setting. MATERIALS AND METHODS: A total of 260 isolates of carbapenem resistant E.coli (n=57), Klebsiella spp. (n=85), Pseudomonas aeruginosa (n=60), and Acinetobacter baumannii (58) isolated from clinical specimens between 2012-2014 at the Christian Medical College, Vellore were included in the study. All the carbapenem resistant isolates were subjected to CarbaNP, MHT and multiplex PCR for detection of carbapenemase genes. RESULTS: CarbaNP was found to be positive in 88% (n=50/57), 81% (n=69/51), 38% (n=23/60) and 81% (n=47/58) for E.coli, Klebsiella spp., P. aeruginosa, and A. baumannii respectively. While in MHT it showed, 89% (n=51/57) and 81 % (n=69/85) for E.coli and Klebsiella spp. respectively. In P.aeruginosa, synergy testing of imipenem plus cloxacillin showed that, 65% of CarbaNP negatives were ampC producers. Overall, the sensitivity and specificity of CarbaNP was found to be 94% and 100 for bla NDM; 77% and 100 % for bla OXA-48 like producers and 81% and 100% for CarbAcinetoNP respectively. CONCLUSION: This observation was more than what was reported in CLSI guidelines. Therefore, it is advisable to evaluate an assay for better laboratory diagnosis at respective regions.

Molecular Characterization of Invasive Carbapenem-Resistant Acinetobacter baumannii from a Tertiary Care Hospital in South India.

INTRODUCTION: Acinetobacter baumannii is an important opportunistic pathogen responsible for causing nosocomial infections. Carbapenems are considered to be the drug of choice to treat infections caused by multidrug-resistant A. baumannii. The prevalent mechanism of carbapenem resistance in A. baumannii is enzymatic degradation by ß-lactamases. Therefore, the aim of the study is to determine the prevalence and distribution of molecular determinants among the clinical isolates of carbapenem-resistant A. baumannii. METHODS: A total of 103 consecutive, non-duplicate carbapenem-resistant A. baumannii isolated from blood and endotracheal aspirates (ETAs) were included in the study. The CarbAcineto NP test was performed for the screening of carbapenemase production. Polymerase chain reaction (PCR) was performed to detect extended spectrum ß-lactamases (ESBLs), metallo-ß-lactamases (MBLs) and oxacillinases (OXAs). PCR was done for the detection of ISAba1 elements, and mapping PCR was performed to identify the position of ISAba1 with respect to the OXA-23-like gene. RESULTS: Among the 103 A. baumannii isolates, 94 were phenotypically identified as carbapenemase producers. blaPER was the most common among the ESBLs. Among MBLs, blaNDM was predominant followed by the blaVIM gene. blaOXA-51 and blaOXA-23 were the most common and present in all 103 isolates. Almost 80% of the isolates had ISAba1 upstream blaOXA-23 gene. CONCLUSION: The blaOXA-23 and blaNDM genes are the most common type of oxacillinases and metallo ß-lactamases, respectively, and contribute to carbapenem resistance in clinical isolates of A. baumannii. The presence of ISAba1 upstream of the blaOXA-23 gene suggests that the insertion element acts as a promoter for its increased expression. FUNDING: Indian Council of Medical Research, New Delhi, India (ref. no. AMR/TF/54/13ECDHII dated 23 October 2013).

Rapid Screening for Carbapenem Resistant Organisms: Current Results and Future Approaches.

Carbapenem producing Enterobacteriaceae (CPE) is a major public health threat. A total of 120 carbapenem resistant E.coli (n=32) and K.pneumoniae (n=88) from blood stream infections were screened for the presence of carbapenem resistant genes KPC, NDM, IMP, VIM, and OXA-48 like using both conventional multiplex PCR and Xpert(®) Carba-R test. Additionally 26 faeces samples were directly screened with Xpert(®) Carba-R test. Of the tested isolates, 40% (n=48) of NDM and 39.2% (n=47/) of OXA-48-like were identified. Co-production of OXA-48 and NDM was seen in 15 (12.5%) isolates. In Xpert(®) Carba-R test, only NDM was identified in 55% (n=66) of tested isolates. Of the tested faeces samples, 12 were identified as carbapenemase producers: nine with NDM, two with the co-production of NDM and VIM and in Klebsiella spp (n=1), NDM and KPC co-production was seen. However, Xpert(®) Carba-R test fails to detect OXA-48 like as compared with multiplex PCR. The sensitivity, specificity, PPV, NPV of Xpert(®) Carba-R test was 100%, 77%, 96% and 100% respectively. Incorporation of OXA-48 like specific sequence in the panel of Xpert(®) Carba-R test may improve its sensitivity and maximize the coverage of assay.