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 ß-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 ß-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.

Neonatal septicaemia caused by diverse clones of Klebsiella pneumoniae & Escherichia coli harbouring blaCTX-M-15.

BACKGROUND & OBJECTIVES: Information about the genetic diversity of the extended-spectrum ß-lactamases (ESBLs) and the clonal relationship of the organisms causing neonatal infections is limited, particularly from India where neonatal mortality is high. This study was undertaken to investigate the molecular epidemiology and risk factors associated with neonatal septicaemia caused by ESBL-producing Klebsiella pneumoniae and Escherichia coli. METHODS: Bloodstream isolates (n=26) of K. pneumoniae (n=10) and E. coli (n=16) from the neonates admitted in a tertiary care hospital in New Delhi during January to May 2008 were characterized. Antimicrobial susceptibility tests were carried out and ESBL production was assessed phenotypically. PCR was carried out for ESBL and ampC genes. Genotyping was performed by pulsed-field gel electrophoresis (PFGE). Conjugation experiments were done to determine the mobility of ESBL genes. Risk factors associated with ESBL-producing K. pneumoniae and E. coli infections were analysed. RESULTS: Resistance rates to most of the antibiotics tested were high, except for imipenem. Among the isolates tested, 60 per cent of K. pneumoniae and 75 per cent of E. coli were ESBL producers. PFGE of the isolates demonstrated a vast diversity of genotypes with no epidemic clones. Despite the clonal diversity, blaCTX-M-15 was detected in 100 per cent of ESBL-positive isolates. The other genes present in ESBL-positive isolates were blaTEM-1, blaSHV-1 , blaSHV-28 , blaSHV-11 , and blaSHV-12 . Class 1 integrons were detected in 7 of 18 ESBL-positive isolates. Moreover, the plasmid carrying blaCTX-M-15 , in E. coli and K. pneumoniae were self transferable. Feeding through an enteral tube was identified as the only risk factor for sepsis by ESBL-producing organisms. INTERPRETATION & CONCLUSIONS: The study emphasises the presence of blaCTX-M-15 in clonally diverse isolates indicating probable horizontal transfer of this gene. The widespread dissemination of CTX-M-15 is of great concern as it further confines the limited therapeutic interventions available for neonates.

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-ß-lactamases (MBLs), extended-spectrum ß-lactamases and AmpCs, were detected phenotypically and/or by PCR followed by DNA sequencing. Transconjugants possessing the bla NDM-1(New Delhi metallo-ß-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. bla NDM-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 bla NDM-1 are the major contributors to carbapenem resistance. The dissemination of the bla NDM-1 is likely linked to Tn125 in diverse clones of the isolates.

A reliable phenotypic assay for detection of ESBLs and AmpCs in MBL-producing gram-negative bacteria with the use of aminophenylboronic acid, dipicolinic acid and cloxacillin.

ESBLs and AmpCs may escape detection when they coexist with metallo-ß-lactamases such as New Delhi Metallo-ß-lactamases-1. In this study a combination disk assay was established using cefotaxime, cefotaxime/clavulanic acid, cefotaxime/clavulanic acid/cloxacillin, cefoxitin and cefoxitin/phenylboronic acid/cloxacillin on Mueller Hinton agar supplemented with dipicolinic acid for determination of ß-lactamases in the presence of NDM-1.

Characterization of novel plasmid-mediated ß-lactamases (SHV-167 and ACT-16) associated with New Delhi metallo-ß-lactamase-1 harbouring isolates from neonates in India.

Neonatal sepsis due to carbapenem-resistant bacteria is difficult to treat due to limited therapeutic options. The detection of the new carbapenemase New Delhi metallo-ß-lactamase-1 (NDM-1) from neonates has further complicated the situation (Roy et al., 2011a). The potent metallo-ß-lactamase NDM-1 efficiently hydrolyses all classes of ß-lactam antibiotics (penicillins, cephalosporins and carbapenems) and is also associated with multiple determinants that enable the bacteria to become resistant to other antibiotic classes (Nordmann et al., 2011). In the presence of NDM-1 other ß-lactamases may go unobserved because of the spectrum of activity of NDM-1 against all ß-lactam antibiotics. Thus, under the canopy of the NDM-1 these ß-lactamases also get the opportunity to spread. This communication reports association of two novel ß-lactamases, SHV-type ß-lactamase (SHV-167) and AmpC-type ß-lactamase (ACT-16), in two NDM-1-carrying Enterobacteriaceae isolated from the blood of two septicaemic neonates admitted to a neonatal intensive care unit.

A five-year experience of carbapenem resistance in Enterobacteriaceae causing neonatal septicaemia: predominance of NDM-1.

Treatment of neonatal sepsis has become a challenge with the emergence of carbapenemase-producing bacteria. This study documents the trend of carbapenem susceptibility in Enterobacteriaceae that caused septicaemia in neonates over a five year period (2007-2011) and the molecular characterisation of Enterobacteriaceae resistant to carbapenems and cephalosporins. Hundred and five Enterobacteriaceae including Escherichia coli (n = 27), Klebsiella pneumoniae (n = 68) and Enterobacter spp. (n = 10) were isolated from blood of septicaemic neonates followed by antibiotic susceptibility tests, determination of MIC values, phenotypic and genotypic detection of ß-lactamases. Carbapenem was the most active antimicrobial tested after tigecycline. CTX-M type was the most prevalent ESBL throughout the period (82%). New Delhi Metallo-ß-lactamase-1 (NDM-1), which is a recent addition to the carbapenemase list, was the only carbapenemase identified in our setting. Fourteen percent of the isolates possessed blaNDM-1. Carbapenem non-susceptibility was first observed in 2007 and it was due to loss of Omp F/Ompk36 in combination with the presence of ESBLs/AmpCs. NDM-1 first emerged in E. coli during 2008; later in 2010, the resistance was detected in K. pneumoniae and E. cloacae isolates. NDM-1-producing isolates were resistant to other broad-spectrum antibiotics and possessed ESBLs, AmpCs, 16S-rRNA methylases, AAC(6')-Ib-cr, bleomycin resistant gene and class 1 integron. Pulsed field gel electrophoresis of the NDM-1-producing isolates indicated that the isolates were clonally diverse. The study also showed that there was a significantly higher incidence of sepsis caused by NDM-1-harbouring isolates in the male sex, in neonates with low birth weight and neonates born at an extramural centre. However, sepsis with NDM-1-harbouring isolates did not result in a higher mortality rate. The study is the first to review the carbapenem resistance patterns in neonatal sepsis over an extended period of time. The study highlights the persistence of ESBLs (CTX-Ms) and the emergence of NDM-1 in Enterobacteriaceae in the unit.