Chaitra Shankar

Molecular Mechanisms of Colistin Resistance in Klebsiella pneumoniae Causing Bacteremia from India—A First Report

Colistin has long been a reserve drug used for the treatment of carbapenem resistant Klebsiella pneumoniae. Carbapenem resistance in K. pneumoniae has been increasing and is as high as 44% in India. Although a reserve agent, with rise in rates of resistance to carbapenems, the usage of colistin has increased over the years leading to slow emergence of resistance. Colistin resistance is mainly mediated by the alteration in the LPS of bacterial outer membrane with the addition of L-Ara4-N and PEtN molecules. These alterations are mediated by mutations in several genes involved in lipidA modifications and most commonly mutations in mgrB gene has been reported. Recently there is emergence of plasmid mediated resistance due to mcr-1 and mcr-2 genes which poses a threat for the rapid global spread. This study aims at characterizing eight colistin resistant K. pneumoniae from bacteremia by whole genome sequencing. Eight K. pneumoniae were isolated from blood culture during 2013 and 2014 at the Department of Clinical Microbiology, Christian Medical College, India. Antimicrobial susceptibility testing was performed and minimum inhibitory concentration (MIC) was determined for colistin and polymyxin B by broth-micro dilution method. Whole genome sequencing was performed using Ion Torrent and the genome of all eight isolates was analyzed. The eight isolates were resistant to all the antimicrobials expect tigecycline. MIC of colistin and polymyxin B were ranged from 4 to 1024 μg/ml and 0.5 to 2048 μg/ml respectively. Multiple mutations were observed in the chromosomal genes involved in lipid A modifications. mcr-1 and mcr-2 gene was absent in all the isolates. The most significant were mutations in mgrB gene. Among the eight isolates, four, three and one were belonged to sequence types ST 231, ST14 and ST147 respectively. Seven isolates had blaOXA−48 like, one co-expressed blaNDM−1 and blaOXA−48 like genes leading to carbapenem resistance. Overall, multiple numbers of alterations have been observed. This includes silent mutations, point mutations, insertions and/or deletions. Mutations in mgrB gene is responsible for resistance to colistin in this study. Due to emergence of resistance to reserve drugs, there is a need for combination therapies for carbapenem resistant K. pneumoniae and colistin must be judiciously used.

Draft Genome Sequences of Three Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Isolates from Bacteremia

ABSTRACT Hypervirulent Klebsiella pneumoniae strains have been increasingly reported worldwide, and there is emergence of carbapenem resistance among them. Here, we report the genome sequences of three carbapenem-resistant hypervirulent K. pneumoniae isolates isolated from bacteremic patients at a tertiary-care center in South India.

Draft Genome Sequence of an Extended-Spectrum-β-Lactamase-Positive Hypervirulent Klebsiella pneumoniae Strain with Novel Sequence Type 2318 Isolated from a Neonate

ABSTRACT Antimicrobial resistance among hypervirulent Klebsiella pneumoniae is increasingly reported. Here, we report the draft genome sequence of a hypervirulent K. pneumoniae strain isolated from a neonate with sepsis belonging to novel sequence type 2318 (ST2318).

Whole genome analysis of hypervirulent Klebsiella pneumoniae isolates from community and hospital acquired bloodstream infection

BackgroundHypervirulent K. pneumoniae (hvKp) causes severe community acquired infections, predominantly in Asia. Though initially isolated from liver abscesses, they are now prevalent among invasive infections such as bacteraemia. There have been no studies reported till date on the prevalence and characterisation of hvKp in India. The objective of this study is to characterise the hypervirulent strains isolated from bacteraemic patients for determination of various virulence genes and resistance genes and also to investigate the difference between healthcare associated and community acquired hvKp with respect to clinical profile, antibiogram, clinical outcome and molecular epidemiology.ResultsSeven isolates that were susceptible to all of the first and second line antimicrobials and phenotypically identified by positive string test were included in the study. They were then confirmed genotypically by presence of rmpA and rmpA2 by PCR. Among the study isolates, four were from patients with healthcare associated infections; none were fatal. All patients with community acquired infection possessed chronic liver disease with fatal outcome. Genes encoding for siderophores such as aerobactin, enterobactin, yersiniabactin, allantoin metabolism and iron uptake were identified by whole genome sequencing. Five isolates belonged to K1 capsular type including one K. quasipneumoniae. None belonged to K2 capsular type. Four isolates belonged to the international clone ST23 among which three were health-care associated and possessed increased virulence genes. Two novel sequence types were identified in the study; K. pneumoniae belonging to ST2319 and K. quasipneumoniae belonging to ST2320. Seventh isolate belonged to ST420.ConclusionThis is the first report on whole genome analysis of hypervirulent K. pneumoniae from India. The novel sequence types described in this study indicate that these strains are evolving and hvKp is now spread across various clonal types. Studies to monitor the prevalence of hvKp is needed since there is a potential for the community acquired isolates to develop multidrug resistance in hospital environment and may pose a major challenge for clinical management.

Clinical and Bacterial Risk Factors for Mortality in Children with Carbapenem- Resistant Enterobacteriaceae Bloodstream Infections in India.

Background: Carbapenem-resistant Enterobacteriaceae (CRE) are an increasing cause of nosocomial infection in hospitalized children worldwide. Few studies have investigated risk factors for mortality in children with CRE bloodstream infection (BSI). Data are particularly scarce in areas where NDM and OXA carbapenemases predominate. Here, we investigate mortality rates, clinical and microbiologic risk factors for mortality in 50 pediatric patients with CRE BSI in India. Methods: Children younger than 17 years old with meropenem-resistant Klebsiella pneumoniae or Escherichia coli isolated from blood culture in 2014 and 2015 were identified from laboratory records. Clinical records were systematically reviewed for each child to establish mortality at 30 days and clinical details. Bacterial isolates were subjected to meropenem E test and multiplex polymerase chain reaction to determine carbapenemase gene. Data were analyzed to establish clinical and bacterial risk factors for mortality. Results: All CRE BSI were hospital-acquired or associated with healthcare. A total of 84% of children had an underlying comorbidity and 46% had a malignancy. K. pneumoniae was the most common bacteria isolated; NDM was the most common carbapenemase gene detected. The mortality rate was 52%. Significant risk factors for mortality included intensive care admission, intubation, inotropic support and respiratory source. Failure to clear bacteremia and a minimum inhibitory concentration > 8 mg/L for the isolate was associated with a statistically significant increase in mortality. Mortality rates were significantly lower when two or more effective drugs were used in combination. Conclusions: CRE BSI affects children with multiple comorbidities and repeated admissions to hospital. The mortality rate is high; combination therapy may be beneficial.

Can minocycline be a carbapenem sparing antibiotic? Current evidence

With the increasing incidence of multidrug-resistant organisms, there is a need for newer antibiotics. However, due to the lack of new antimicrobial agents, it is necessary to re-evaluate the older agents like minocycline which is a second-line antimicrobial agent. In this study, minocycline susceptibility testing was performed for 693 Escherichia coli, 316 Klebsiella spp. and 89 Acinetobacter spp. Among extended spectrum beta-lactamase producing E. coli and Klebsiella spp. percentage susceptibility to minocycline were 76 and 85, respectively. Among the carbapenem resistant E. coli, Klebsiella spp. and Acinetobacter spp. minocycline susceptibility were 52%, 55% and 42%, respectively. Based on the susceptibility profile, minocycline can be considered for treatment of infections by multidrug-resistant organisms.

Carbapenem resistant Klebsiella pneumoniae isolated from bloodstream infection: Indian experience.

Abstract Increased incidence of multidrug resistant (MDR) Gram negative infection has resulted in high rates of morbidity and mortality. Klebsiella pneumoniae is one of the commonest MDR pathogens causing bacteraemia with limited therapeutic options such as colistin and tigecycline. Present study focused on molecular characterisation of MDR K. pneumoniae from bloodstream infection and their clinical outcome. A total of 115 K. pneumoniae from January 2015 to September 2016 were included in the study which comprised of phenotypically identified ESBL and carbapenem resistant (CR) isolates. Multiplex PCR was performed for detection of resistance genes encoding β-lactam resistance. This includes blaSHV, blaTEM, blaVEB, blaPER, blaCTX-M, blaDHA, blaCIT, blaFOX, blaACC, blaACT, blaNDM, blaOXA48-like, blaVIM and blaKPC. Co-expression of blaSHV, blaTEM and blaCTX-M was predominant with 64% (74/115) prevalence. CTX-M-1 was the variant produced by all the isolates producing CTX-M. AmpC was uncommon, seen in 5% of the isolates (6/115). Among the carbapenemases co-expression of blaNDM and blaOXA48-like was observed in 28% (32/115) and blaNDM in 19% (22/115) and blaOXA48-like in 13% (15/115). blaKPC was absent. Overall mortality was observed to be 57% (64/113) and mortality among CR K. pneumoniae (Kp) was 68% (50/73). The antibiotics that were administered for treatment of CRKp were colistin in 90% (66/73) and tigecycline in 7% (5/73) and in 99% combined with meropenem (72/73). Prevalence of community acquired and nosocomial infections were 5% (4/73) and 95% (69/73) respectively among CRKp. Minocycline and meropenem susceptibilities were comparable and hence minocycline can be a carbapenem sparing agent. The resistance to β-lactam antibiotics is steadily increasing and are plasmid mediated, their containment in healthcare setting is a challenge.

Detection of chromosomal and plasmid mediated mechanisms conferring colistin resistance in Escherichia coli and Klebsiella pneumoniae from Indian food samples

OBJECTIVES Numerous previous publications on the detection of bacterial isolates harbouring the mcr-1 gene from animals and humans strongly suggest an underlying route of transmission of colistin resistance via the food chain. The aim of this study was to investigate the presence of colistin-resistant (Col-R) bacteria in Indian food samples and to identify the underlying mechanisms conferring colistin resistance. METHODS Raw food material, including poultry meat, mutton meat, fish, fruit and vegetables, collected from food outlets in Chennai, India, were processed to identify Col-R bacteria using eosin methylene blue agar supplemented with colistin. Colistin minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. PCR for the mcr-1 and mcr-3 genes was performed on Col-R Escherichia coli and Klebsiella pneumoniae isolates. Mutations in the mgrB gene were analysed in K. pneumoniae isolates. One representative mcr-1-positive E. coli was subjected to whole-genome sequencing. RESULTS Of 110 food samples tested, 51 (46.4%) were positive for non-intrinsic Col-R Gram-negative bacteria. Three E. coli isolates were found to harbour mcr-1, whereas none were positive for mcr-3. Ten K. pneumoniae isolates had alterations in mgrB, with mutations in four and insertional inactivation in six. CONCLUSION The presence of Col-R bacteria and the mcr-1 gene in raw food samples further complicates the antimicrobial resistance scenario in India. To the best of our knowledge, this is the first report in the global literature on mgrB mutation and its insertional inactivation conferring Col-R in K. pneumoniae from food samples.