SummaryBackgroundGram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK.MethodsEnterobacteriaceae isolates were studied from two major centres in India—Chennai (south India), Haryana (north India)—and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene blaNDM-1 was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan.FindingsWe identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries.InterpretationThe potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.FundingEuropean Union, Wellcome Trust, and Wyeth.

The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently become a topic of intense scrutiny. Here, we characterized the behavior of MC/Mph in cellular infiltrates, with emphasis on their spatial organization and localization in newly formed microvessels. To this end, we studied MC/Mph migration and assembly in basic fibroblast growth factor-supplemented Matrigel plugs placed in transgenic Tie2-beta-galactosidase mice for up to 4 weeks. In these plugs, along with Nile Red-positive adipocytes, we found MC/Mph distributed in cell cords, also containing various mature and progenitor tissue cells; and functional Tie2-positive or -negative microvessels embedded in bundles of fibrillar collagen surrounded by F4/80-positive MC/Mph. At earlier stages of infiltration, we found tubular destruction of the matrix (tunnels) and MC/Mph-lined capillary-like structures occasionally containing erythrocytes, indicating their propensity for endothelial trans-differentiation. We also analyzed in vitro the MCP-1-induced chemotactic migration of fluorescently labeled peritoneal MC/Mph incorporated in Matrigel-containing fluorescent protease substrates. Many of these MC/Mph produced MMP-12- and TIMP-1-dependent tunnels coupled with acquisition of a lumen. In conclusion, long-term implantation of Matrigel plugs qualifies as a novel experimental model of tissue regeneration, in which neovascularization intimately couples with fibrosis and organogenesis and in which cells of MC/Mph phenotype play a key structural role.

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Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to neovascularization, but their nature and significance remains elusive. Based on the emerging evidence implicating a role for monocytes and macrophages (MC/MPH) in vasculogenesis, we hypothesized that MC/MPH also can form linear or branched columns, facilitating the co-migration and the spatial arrangement of other cell types. To test this hypothesis, we studied the distribution of MC/MPH effected by chemotactic migration in novel in vitro and in vivo models of development. We induced transversal and lateral migration of THP-1 monocytoid cells in Matrigel in vitro. The effect of this process on co-localization of other micro-objects was assessed using erythrocytes and micron-sized plastic beads. In vivo, we analyzed MC/MPH infiltration in subcutaneously implanted Matrigel plugs containing angiogenic factors and across a microporous filter comprising the wall of a chamber filled with Matrigel, also placed subcutaneously in mice. In vitro, we found that migrating THP-1 cells induced the lasting degradation of Matrigel and produced cell columns, a process amplified by monocyte chemoattractant protein-1 (MCP-1). We also report the co-localization of erythrocytes with THP-1 cells in cell columns. Endothelium-free tunnels containing MC/MPH, neutrophils, or erythrocytes were also observed in the Matrigel-filled chambers. In free subcutaneous Matrigel plugs, we found MC/MPH-based columns harboring isolated Tie-2+ cells (a marker of endothelial progenitor phenotype), as well as fibroblasts, dendritic cells, and adypocytes. Many of these cell columns displayed conspicuous branching. Our data demonstrate formation of branched MC/MPH cell columns in vitro and in vivo, a previously unrecognized pattern of penetration of extracellular matrices by inflammatory cells. Thus, monocytes and macrophages influence the distribution of neovessels as well as their branching points. These cells are the "architects of development," assisting organogenesis, tumorigenesis, and wound healing by patterning the tissular space.

IntroductionThe present study attempts to examine the microbial profile and antibiotic susceptibility of diabetic foot infections in the intensive care unit of a tertiary referral centre for diabetic foot. As part of the study, we also attempted to find the prevalence of blaNDM-like gene among carbapenem-resistant gram negative infections.MethodologyA prospective study of 261 patients with diabetic foot infections was performed during the period between January 2014 and June 2014.ResultsA total of 289 isolates were obtained from 178 tissue samples from 261 patients, 156 (59.7%) males and 105 (40.2%) females, with a mean age of 58 years (−15 years), having diabetic foot infection. No growth was seen in thirty eight (17.6%) tissue samples. Out of the total samples, 44.3% were monomicrobial and 55.7% were polymicrobial. Gram negative pathogens were predominant (58.5%). Seven of the total isolates were fungal; 0.7% showed pure fungal growth and 1.7% were mixed, grown along with some bacteria. The most frequently isolated bacteria were Staphylococcus aureus (26.9%), followed by Pseudomonas aeruginosa (20.9%). Of the 58.5% gram negative pathogens, 16.5% were Enterobacteriaceae resistant to carbapenems. Among these isolates, 4 (25%) were positive for blaNDM-like gene. Among the rest, 18.6% were carbapenem-resistant Pseudomonas, among which 4 (36.3%) were blaNDM. Among the Staphylococci, 23.7% were methicillin-resistant Staphylococcus aureus.ConclusionsOur results support the recent view that gram negative organisms, depending on the geographical location, may be predominant in DFIs. There is an increase in multidrug-resistant pathogens, especially carbapenem resistance and this is creeping rapidly. We need to be more judicious while using empiric antibiotics.

kTo determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The bla NDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: bla NDM-1 in 2 isolates, bla NDM-5 in 7 isolates, and bla NDM-7 in 5 isolates. The bla CTX-M gene was detected in 112 isolates (66.3%): bla CTX-M-15 in 108 isolates and bla CTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the bla NDM -positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

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