Sulagna Basu

Transmission of imipenem resistance determinants during the course of an outbreak of NDM-1 Escherichia coli in a sick newborn care unit

OBJECTIVES This study reports a cluster of septicaemic newborns with imipenem-resistant Escherichia coli in the blood and delineates the possible mechanisms of transmission of imipenem resistance with respect to the New Delhi metallo-β-lactamase (NDM-1) gene. METHODS During a point prevalence survey, attempts were made to isolate Gram-negative bacilli (GNB) from the environment of a sick newborn care unit (SNCU) and body sites of neonates. Subsequently, four fresh neonates admitted to the SNCU developed sepsis with E. coli. E. coli isolates from body sites and blood of the newborns were analysed in terms of clonality, carbapenemases, integrons, virulence factors, porins and transmissibility. RESULTS During the survey, both imipenem-resistant and imipenem-susceptible E. coli were isolated from the body sites of neonates, but none from the environment. None of these neonates developed sepsis. The freshly admitted septicaemic neonates had imipenem-resistant E. coli in their blood, which were similar to the imipenem-susceptible E. coli obtained from the body sites (during the survey) in terms of clonality, phylogroup, virulence and other resistance genes, except possession of bla(NDM-1). Imipenem-resistant E. coli from blood and body sites were not clonal, though both had bla(NDM-1). Besides E. coli, other GNB isolated from the environment and body sites also harboured bla(NDM-1). Imipenem-resistant and imipenem-susceptible E. coli from the blood and body sites respectively, possessed a novel AmpC β-lactamase, bla(CMY-59). The plasmid carrying bla(NDM-1) was transferable. CONCLUSIONS The time frame of isolation and clonal identity indicated a possible transfer of bla(NDM-1) from imipenem-resistant GNB to the imipenem-susceptible E. coli, which subsequently caused septicaemia. This establishes the promiscuous nature of bla(NDM-1) and emphasizes the need for the early recognition of similar isolates.

Sepsis in neonates due to imipenem-resistant Klebsiella pneumoniae producing NDM-1 in India

Sir, Treatment of neonatal infections is becoming increasingly difficult due to multidrug-resistant organisms. Carbapenems are the last resort for the treatment of severe infections, however, the emergence of carbapenemases in Enterobacteriaceae has left the clinician cornered with very few options. Recently a new carbapenemase designated New Delhi metallo-blactamase (NDM-1) was identified in Klebsiella pneumoniae. Most reported cases of infection with NDM-1 producers have involved adult patients. This communication reports the presence of blaNDM-1 in two isolates of K. pneumoniae from neonates admitted to a neonatal intensive care unit (NICU) at a tertiary care centre in India. The first neonate was clinically septic. Imipenem-resistant K. pneumoniae (Kp-1) was isolated from an endotracheal aspirate, but blood culture was negative. The patient was started on colistin, however, the parents removed the child from the hospital against medical advice and the patient was lost to follow-up. The other neonate was born at a different hospital and was admitted to the NICU with suspected sepsis. Blood culture yielded imipenem-resistant K. pneumoniae (Kp-2). A combination of colistin and minocycline was given for a period of 10 days, after which the patient recovered and was discharged. The identity of the isolates was confirmed by an ID 32 E kit (bioMérieux, Marcy l’Étoile, France). A disc diffusion test using antibiotics (BD Diagnostics, Franklin Lakes, NJ, USA) was performed according to CLSI guidelines. MICs were determined using Etest (AB Biodisk, Solna, Sweden). MICs were also determined with 40 mg/L phenylalanine arginine b-naphthylamide (PAbN) (Sigma-Aldrich, St Louis, MO, USA). Testing with the cephalosporin/clavulanic acid combination disc test, modified Hodge test and imipenem/EDTA double-disc combination tests was also undertaken. Both isolates were resistant to a range of antibiotics, including b-lactams, quinolones and aminoglycosides. Kp-1 was susceptible only to colistin and tigecycline, but Kp-2 was susceptible to colistin, tigecycline, tetracycline, minocycline and doxycycline (Table 1). Both isolates showed phenotypic evidence of carbapenemase, metallo-b-lactamase (MBL) and extended-spectrum b-lactamase (ESBL) production. Augmentation of the zone was noted in both isolates with ceftazidime, but not cefotaxime. Investigations of production of AmpC using cefoxitin, cefoxitin with 3-aminophenylboronic acid (APB) (Sigma) and cefoxitin plus APB containing EDTA (Sigma) were negative. PFGE performed following PulseNet standardized procedures with XbaI (http://www.cdc.gov/pulsenet/protocols.htm) demonstrated that the two isolates were clonally distinct (Figure S1, available as Supplementary data at JAC Online). The MICs of imipenem were .32 mg/L and not affected by the presence of PAbN, indicating that efflux did not contribute to carbapenem resistance. To elucidate the mechanism of carbapenem resistance, PCR for blaKPC, 5 blaVIM,IMP,SPM-1,GIM-1,SIM-1 2 and blaOXA-23,OXA-24,OXA-48,OXA-58 6 was carried out as previously described. Detection of NDM-1 was performed by PCR with Research letters

Multi-drug resistant gram negative bacilli causing early neonatal sepsis in India

Objective To study the organisms causing early and late onset neonatal sepsis, with special reference to multi-drug resistant gram negative bacilli, at two neonatal units (one urban, one rural) in India. Methods Prospective surveillance study. Results There were 159 episodes of sepsis (81 urban and 77 rural) affecting 158 babies. Gram negative bacilli caused 117 infections (68%) and predominated at both centres in both early and late sepsis. Klebsiella pneumoniae was the commonest organism, causing 61 infections (38.3%). In early sepsis (0–2 days), non-fermenting gram negative bacilli caused 42.1% of infections at the urban centre; there were no cases of early Group B Streptococcus sepsis. Late onset sepsis was mainly caused by gram negative bacilli at both centres. Multi-drug resistance of over 80% of early-onset gram negative organisms to ampicillin, third generation cephalosporins and gentamicin indicates that these multi-resistant organisms are almost certainly circulating widely in the community. The overall mortality from early sepsis was 27.3% (9 of 33) and from late sepsis was 26.2% (33 of 126). Gram negative bacilli caused all deaths from early sepsis and 87.5% of deaths from late sepsis. Conclusion This study shows that multi-drug resistant gram negative bacilli are a major cause of early and late neonatal sepsis in India and are almost certainly widespread in the community.

Tigecycline susceptibility in Klebsiella pneumoniae and Escherichia coli causing neonatal septicaemia (2007–10) and role of an efflux pump in tigecycline non-susceptibility

OBJECTIVES To investigate the trend of tigecycline susceptibility and mechanisms behind tigecycline non-susceptibility in Klebsiella pneumoniae and Escherichia coli isolates causing neonatal septicaemia (2007-10). METHODS MICs of tigecycline for the isolates were determined. The isolates were evaluated for β-lactamases and carbapenemases. Molecular typing of the tigecycline-resistant isolates was performed. Expression of efflux pump genes (acrA, acrB and tolC) and regulators (soxS and ramA) was examined by real-time RT-PCR and western blotting. Sequencing of the ramA and ramR genes was carried out to identify mutations within these genes. RESULTS Tigecycline susceptibility was evaluated in all K. pneumoniae (n = 57) and E. coli (n = 19) blood isolates. The prevalence of extended-spectrum β-lactamase (ESBL)-producing organisms was high, but tigecycline non-susceptibility remained low in these isolates. Though MIC values of tigecycline remained in the susceptible range, there was a 2-fold increase in the value of MIC90 from 2007 to 2010. Over the 4 year period K. pneumoniae showed higher MIC values of tigecycline in comparison with E. coli. Tigecycline non-susceptibility was not observed among carbapenem-resistant isolates. Only two ESBL-producing clonally distinct K. pneumoniae isolates showed tigecycline resistance with overexpression of ramA and the AcrAB-TolC pump. No mutations were present within the ramA and ramR genes that might enhance the expression of the pump. CONCLUSIONS The study showed for the first time the trend of tigecycline susceptibility in E. coli and K. pneumoniae causing neonatal septicaemia. Tigecycline still has potent antimicrobial effects against most ESBL- or carbapenemase-producing K. pneumoniae and E. coli, but the increasing MIC values make it essential to be vigilant.

Colonization of the gut with Gram-negative bacilli, its association with neonatal sepsis and its clinical relevance in a developing country.

This study examined the pattern of colonization of the neonatal gut by aerobic Gram-negative bacilli (GNB) and evaluated the association between gut colonization and sepsis in the developing world. This deserves attention because of the high incidence of sepsis and the differences in hygienic environments in developing countries compared with the developed world. The study was carried out on neonates in a tertiary-care government hospital. Serial gut samples were analysed (gastric aspirates and stool samples) for GNB. Blood samples of cases showing clinical signs of sepsis were also analysed for septic screening and culture positivity. Antibiograms, serotyping and PFGE were carried out to evaluate the relatedness of the gut and blood isolates. A diverse array of GNB was isolated from the gut of the neonates, Klebsiella pneumoniae being most common, followed by Escherichia coli. The rate of isolation of GNB was consistently higher in stool samples compared with gastric aspirate samples. Colonization was influenced by a stay in the neonatal intensive care unit and by the prolonged use of a feeding tube. GNB were the cause of sepsis in the majority of cases, with K. pneumoniae being the most frequently isolated GNB from the blood. Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae and Burkholderia cepacia were the other GNB recovered from the blood of the neonates. Neonates with GNB in the gut had a higher incidence of clinical sepsis than those without. In 50 % of cases, the genotypes of the organisms found in the blood were indistinguishable from their gut counterpart. These results show that the neonatal gut is colonized with a diverse array of GNB, and an association between gut colonization and neonatal sepsis was observed.

Polyethylene glycol-stabilized sulphur nanoparticles: an effective antimicrobial agent against multidrug-resistant bacteria

OBJECTIVES To elucidate the antibacterial efficacy of chemically synthesized and custom-made sulphur nanoparticles (SNPs) of two different sizes and surface modifications against a number of multidrug-resistant Gram-negative bacilli (GNB) harbouring the New Delhi metallo-β-lactamase 1 enzyme (NDM-1). METHODS Antimicrobial susceptibility of the isolates was determined. The strains were evaluated for the presence of carbapenemases, β-lactamases, 16S rRNA methylases and integrons. Chemically synthesized, polyethylene-glycol (PEG)-stabilized SNPs of 10 nm and custom-made non-capped SNPs of 60 nm were physicochemically characterized and evaluated for their antibacterial efficacy against multidrug-resistant GNB using the agar dilution method (ADM) and the broth microdilution method (BMD). The cytotoxicity of the chemically synthesized SNPs was evaluated with a human-derived hepatoma (HepG2) cell line using a WST-1 assay kit. RESULTS All isolates were multidrug-resistant and possessed NDM-1 along with other β-lactamases, 16S rRNA methylases and integron 1. Chemically synthesized PEGylated SNPs showed a bactericidal effect against all tested strains at a concentration between 9.41 and 18.82 mg/L using BMD. The ADM data revealed that SNPs had uniform MICs (18.82 mg/L) for all tested strains. On the other hand, custom-made SNPs failed to impart any antibacterial effect at the equivalent concentrations of chemically synthesized SNPs. The WST-1 assay revealed no significant cytotoxicity of the PEGylated SNPs even at the highest concentration (94.08 mg/L). CONCLUSIONS To the best of our knowledge, this is the first attempted study to show the effectiveness of nanoparticles against multidrug-resistant GNB harbouring NDM-1.

Investigation of antimicrobial physiology of orthorhombic and monoclinic nanoallotropes of sulfur at the interface of transcriptome and metabolome

Nanosized elemental sulfur (ES) is already reported to exert superior antimicrobial efficacy than micron-sized ES, which encourages their use in drugs and therapeutics. The aim of the present study is to explore the possible route and mode of antimicrobial action of orthorhombic (α-SNPs) and monoclinic (β-SNPs) allotropes of sulfur, respectively, at their nano-dimensions. The antimicrobial efficacy of α- and β-SNPs was determined against both the conventionally ES-resistant and ES-susceptible fungi and bacteria. Both the SNPs inhibited the microbial growth, irrespective of their resistance profile to ES and caused significant deformities on the microbial cell surfaces. However, the extent of antimicrobial efficacy was found to be optimum for α-SNPs, which can be attributed to their size, shape, and surface modification. Subsequent transcript profiling, metabolite profiling, and enzymatic analyses revealed that α- and β-SNPs impaired a cluster of mitochondrial enzymes involved in cellular respiration and oxidative phosphorylation. ES and SNPs stress were found to elicit the NADPH-dependent glutathione reductase mediated ES-detoxification response in fungi and caused them to undertake the glyoxylate shunt in favor of energy conservation. A simultaneous study was also undertaken to assess the biocompatible or bio-adverse properties of SNPs in terms of their cytotoxic and genotoxic effects against the human derived lung fibroblast cell line (MRC-5). The present study hence explores the antimicrobial physiology of two novel functional materials and demonstrates their compatibility as a future putative antimicrobial drug.

Carbapenem Resistance in Acinetobacter baumannii and Other Acinetobacter spp. Causing Neonatal Sepsis: Focus on NDM-1 and Its Linkage to ISAba125

Carbapenem-resistant determinants and their surrounding genetic structure were studied in Acinetobacter spp. from neonatal sepsis cases collected over 7 years at a tertiary care hospital. Acinetobacter spp. (n = 68) were identified by ARDRA followed by susceptibility tests. Oxacillinases, metallo-β-lactamases (MBLs), extended-spectrum β-lactamases and AmpCs, were detected phenotypically and/or by PCR followed by DNA sequencing. Transconjugants possessing the blaNDM−1(New Delhi metallo-β-lactamase) underwent further analysis for plasmids, integrons and associated genes. Genetic environment of the carbapenemases were studied by PCR mapping and DNA sequencing. Multivariate logistic regression was used to identify risk factors for sepsis caused by NDM-1-harboring organisms. A. baumannii (72%) was the predominant species followed by A. calcoaceticus (10%), A. lwoffii (6%), A. nosocomialis (3%), A. junni (3%), A. variabilis (3%), A. haemolyticus (2%), and 14TU (2%). Fifty six percent of the isolates were meropenem-resistant. Oxacillinases present were OXA-23-like, OXA-58-like and OXA-51-like, predominately in A. baumannii. NDM-1 was the dominant MBL (22%) across different Acinetobacter spp. Isolates harboring NDM-1 also possessed bla(VIM−2, PER−1, VEB−2, CTX−M−15), armA, aac(6′)Ib, aac(6′)Ib-cr genes. blaNDM−1was organized in a composite transposon between two copies of ISAba125 in the isolates irrespective of the species. Further, OXA-23-like gene and OXA-58-like genes were linked with ISAba1 and ISAba3 respectively. Isolates were clonally diverse. Integrons were variable in sequence but not associated with carbapenem resistance. Most commonly found genes in the 5′ and 3′conserved segment were aminoglycoside resistance genes (aadB, aadA2, aac4′), non-enzymatic chloramphenicol resistance gene (cmlA1g) and ADP-ribosylation genes (arr2, arr3). Outborn neonates had a significantly higher incidence of sepsis due to NDM-1 harboring isolates than their inborn counterparts. This study demonstrates the significance of both A. baumannii and other species of Acinetobacter in cases of neonatal sepsis over an extended period. Oxacillinases and blaNDM−1 are the major contributors to carbapenem resistance. The dissemination of the blaNDM−1 is likely linked to Tn125 in diverse clones of the isolates.

Fatbodies and spermatogenesis in South Indian green frog Rana hexadactyla Lesson

Abstract The role of fatbodies in the normal gonadal function of the male frog Rana hexadactyla has been studied. Present experiments have revealed that the hypothalamus exercises a control through hypophysis over the mobilization of fatbody contests which in turn control the testicular activity. The metabolites of the fatbody contents are released into the testis through the direct blood supply between the two organs which has been established by earlier workers. The role of A, and B2 cells of the pars distalis in the mobilization of fatbody contents has already been reported. It has been found in the present study that gonadrotrophins could not stimulate gonads in the absence of fatbodies. In view of these observations, a possible modus operandi has been discussed.

Multi-drug-resistant, non-fermenting, gram-negative bacilli in neonatal sepsis in Kolkata, India: a 4-year study

Abstract Background: Non-fermenting gram-negative bacilli (NFGNB) are an emerging problem in neonatal sepsis. A major concern is multi-drug resistance which severely limits treatment options. Aims and Objectives: A retrospective observational study was conducted to analyse the role of non-fermenters in neonatal sepsis over a 4-year period, the factors leading to this trend and the pattern of antibiotic resistance. Methods: Demographic and clinical data were collected for all neonates with blood culture-positive sepsis during the study period, January 2007 to December 2010. Results: Blood cultures were positive in 186 (13%) of 1402 neonates, in 44 (32·1%) of whom the cause was NFGNB. Acinetobacter spp was the most common organism (n = 30). Infection by NFGNB showed a steady increase (P<0·0001), and was fairly evenly distributed between early- and late-onset sepsis. The infection rate was significantly higher in inborn neonates (P = 0·04) and those delivered vaginally (P = 0·002). Multi-drug resistance (MDR) occurred in 50% and carbapenem resistance in 30% of Acinetobacter spp isolates. In five cases there was panresistance of Acinetobacter spp to all antibiotics tested. Conclusion: The trend of increasing numbers of cases of NFGNB in neonatal sepsis compounded by MDR is of great concern. It is necessary to administer antibiotics judiciously, strengthen surveillance and laboratory services in neonatal intensive care units, and re-evaluate treatment guidelines for management of infection by these organisms.