Subhasree Roy

Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India

As the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 x 10(2) cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.

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

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.

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.

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.

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.