Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer “average days alive” for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

Recent findings demonstrate the origin of the plasmid-mediated colistin resistance gene mcr-3 from aeromonads. The present study aimed to screen for plasmid-mediated colistin resistance among 30 clinical multidrug-resistant (MDR) Aeromonas spp. PCR was used to screen for the presence of mcr-1 , mcr-2 , mcr-3 and mcr-4 , which revealed mcr-3 in a colistin-susceptible isolate (FC951). All other isolates were negative for mcr . Sequencing of FC951 revealed that the mcr-3 ( mcr-3.30 ) identified was different from previously reported variants and had 95.62 and 95.28 % nucleotide similarity with mcr-3.3 and mcr-3.10 . Hybrid assembly using IonTorrent and MinION reads revealed structural genetic information for mcr-3.30 with an insertion of IS As18 within the gene. Due to this, mcr-3.30 was non-expressive, which makes FC951 susceptible to colistin. Further, in silico sequence and protein structural analysis confirmed the new variant. To the best of our knowledge, this is the first report on a novel mcr-3 variant from India. The significant role of mcr -like genes in different Aeromonas species remains unknown and requires additional investigation to obtains insights into the mechanism of colistin resistance.

Shigella is the major cause of bacillary dysentery worldwide, especially in developing countries. There are several virulence factors essential for the organism to be virulent which are generally present in the virulence plasmid and on chromosomal pathogenicity islands. The present study was undertaken to determine the virulence gene profile of Shigella spp isolated from a clinical specimen and to study their significant association with common clinical symptoms and antimicrobial resistance. Sixty Shigella whole genome sequences, including 22 S. flexneri, 14 S. sonnei, 17 S. boydii and 7 S. dysenteriae were analyzed for the presence of virulence genes. The gene found predominantly in this study were ipaH (90%) followed by sigA (83%), and lpfA (78%) respectively. The virulence genes were significantly higher in S. flexneri, particularly in serotype 2 compared to S. sonnei. Interestingly, a significant association was observed between sigA gene and fever whereas sepA and sigA were found to be associated with diarrhea. Among the studied Shigella isolates, the presence of virulence genes was found higher in isolates resistant to more than three antibiotic classes. The present work revealed the varying incidence of virulence determinants among different Shigella serogroups and shows their contribution to disease severity.

Shigella is ranked as the second leading cause of diarrheal disease worldwide. Though infection occurs in people of all ages, most of the disease burden constitutes among the children less than 5 years in low and middle income countries. Recent increasing incidence of drug resistant strains make this as a priority pathogen under the antimicrobial resistance surveillance by WHO. Despite this, only limited genomic studies on drug resistant Shigella exists. Here we report the first complete genome of clinical S. flexneri serotype 2a and S. sonnei strains using a hybrid approach of both long-read MinION (Oxford Nanopore Technologies) and short-read Ion Torrent 400 bp sequencing platforms. The utilization of this novel approach in the present study helped to identify the complete plasmid sequence of pSS1653 with structural genetic information of AMR genes such as sulII, tetA, tetR, aph(6)-Id and aph(3′’)-Ib. Identification of AMR genes in mobile elements in this human-restricted enteric pathogen is a potential threat for dissemination to other gut pathogens. The information on Shigella at genome level could help us to understand the genome dynamics of existing and emerging resistant clones.

Introduction. Bacterial dysentery is one of the greatest causes of morbidity and mortality worldwide. Campylobacter spp. and diarrhoeagenic Escherichia coli (DEC) are recognised as the most common causes of bacterial enteritis in developing countries including India. Hypothesis/Gap statement. Rapid and accurate identification of dysentery causing organisms using molecular methods is essential for better disease management, epidemiology and outbreak investigations. Aim. In view of the limited information available on the dysentery causing agents like Campylobacter spp., enterohemorrhagic E. coli (EHEC)/enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC)/ Shigella in India, this study was undertaken to investigate the presence of these pathogens in human and poultry stool samples by molecular methods. Methodology. In total, 400 human stool samples and 128 poultry samples were studied. Microaerophilic culture along with real-time multiplex PCR with the targets specific to the genus Campylobacter , Campylobacter jejuni , Campylobacter coli , EHEC, EPEC and EIEC/ Shigella was performed. Further species confirmation was done using MALDI-TOF MS. Results. On microaerophilic culture, C. coli was isolated in one human sample and two C. jejuni and one C. fetus in poultry samples. On PCR analysis, among human stool samples, typical EPEC (42%) was predominantly seen followed by Campylobacter spp. (19%) and EIEC/ Shigella (10%). In contrast, Campylobacter spp. (41%) was predominant in poultry samples, followed by typical EPEC (26%) and EIEC/ Shigella (9%). Poly-infections with Campylobacter spp. and DEC were also observed among both sources. Conclusion. The present study documented the increased prevalence of Campylobacter spp. in humans compared with the results of previous studies from India. Typical EPEC was found to be predominant in children less than 5 years of age in this study. The high prevalence of coinfections in the current study indicates that a multiple aetiology of diarrhoea is common in our settings.