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Summary Background Enterobacter cloacae complex contains nosocomial pathogens responsible for infection outbreaks. Identification at the species level within the E cloacae complex remains difficult. Using whole genome sequencing, our aim was to decipher the transmission routes that led to the death of six of seven neonates who had bacteraemia caused by E cloacae complex isolates in a neonatal intensive care unit (NICU) over a 13 month period. Methods In this cohort study, E cloacae complex isolates were taken from 186 newborns in an NICU: 14 were clinical samples (eg, blood cultures), 728 rectal swab samples, and 38 environmental samples (20 from siphons, 18 from incubators, and one from a mattress). The samples were analysed to decipher the possible role of manual cross transmission or environmental source in an outbreak of fatal septic shocks related to E cloacae complex bacteraemia. After the replacement of the incubators suspected to be the reservoir of some outbreak-related isolates on Feb 1, 2018, E cloacae complex strains were screened again for 10 months (503 rectal swab samples from 163 newborns). The 71 E cloacae complex isolates recovered from screening, clinical, and environmental samples across both study periods were compared by whole genome sequencing. The pathogenicity of E cloacae complex isolates responsible for fatal septic shocks was assessed using a Galleria mellonella in-vivo model. Findings From Dec 9, 2016, to Jan 31, 2018, 249 (34%) of 728 rectal swab samples were positive for E cloacae complex, with 66 (35%) of 186 newborns colonised. E cloacae complex were also recovered from four (20%) of 20 siphons and 11 (61%) of 18 incubators. During this period, whole genome sequencing identified that the isolates responsible for the six fatal septic shocks were all Enterobacter bugandensis. A G mellonella infection model showed a higher virulence of E bugandensis. Genomic analysis highlighted the role of incubators as a long-term reservoir and source of cross contamination, leading to their replacement on Feb 1, 2018. Following incubator replacement, a 10-month follow-up investigation identified a physiological gut colonisation with polyclonal E cloacae complex in 52 (34%) of 163 neonates within a median of 9 days (5–14), but no E cloacae complex-related septic shocks. Interpretation Despite around 30% of neonates being physiologically colonised with E cloacae complex, fatal sepsis was systematically linked with bacteraemia caused by E bugandensis. Our findings highlight the need for accurate identification methods to detect the hypervirulent species within the E cloacae complex recovered in neonates. Funding Assistance Publique–Hopitaux de Paris.

As carbapenemase-producing Enterobacteriaceae (CPE) and extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The ELITe InGenius is a fully automated sample-to-result system designed for the extraction and detection by multiplex real-time polymerase chain reaction of carbapenemases KPC, NDM, VIM, IMP, and OXA-48–like variants and CTX-M group 1 and 9-producers from diverse sample matrices such as colonies, positive blood cultures, and rectal swabs. CRE and ESBL ELITe MGB® kits were evaluated on 153 cultured colonies of enterobacterial isolates with characterized β-lactamase content, on 30 spiked blood cultures, and the CRE kit was also evaluated on 53 clinical rectal swabs collected prospectively during a 3-month period and 10 spiked rectal swabs. CRE ELITe MGB® kit’s performances reached 100% sensitivity and 100% specificity, while for the ESBL ELITe kit, 100% sensitivity and 96.6% specificity were observed, with a sample to result of less than 3 h and a total percentage of agreement with expected results of 99.6% (255/256).

A multilocus sequence typing (MLST) scheme including 7 housekeeping genes was used to evaluate whether the current spread of multidrug-resistant Enterobacter cloacae isolates worldwide might be associated to specific successful clones. Fifty E. cloacae clinical isolates of worldwide origin, with various β-lactamase content, and recovered at different periods of time were studied. Forty-four sequence types were identified, highlighting a high clonal diversity with 3 main lineages. This study revealed that a precise identification of the isolates by sequencing of the chromosomal ampC gene of E. cloacae would provide a significant added value to improve the reliability of the MLST scheme.

An OXA-181 producing Escherichia coli isolate that went recurrently undetected directly from rectal swab sample using Xpert® Carba-R, was successfully detected using ertapenem supplemented broth enrichment followed by culture-based method. Our data suggest that implementation of culture-based methods plus enrichment might be crucial for the efficient screening of patients considered to be at “high-risk” of colonization with carbapenemase producers and who are colonized at low level.

The recently modified CHROMagar Acinetobacter medium was evaluated for detection of carbapenemase-producing Acinetobacter baumannii from spiked stools. A total of 45 Acinetobacter spp. isolates were tested. The CHROMagar Acinetobacter medium had a high sensitivity of 86.5% and a specificity of 75%. This medium is likely to be most useful for controlling outbreaks and in endemic situations.

Introduction Carbapenemase-producing enterobacteriaceae (CPE) and non fermenters (CPNF; pseudomonadaceae and Acinetobacter sp.) have been increasingly reported worldwide. The most clinically relevant carbapenemases belong either to Ambler class A (KPC-type), Ambler class B, i-e metallo-b-lactamases (MBLs such as IMP-, VIM- and NDM-types) or Ambler Class D (OXA-48-like in enterobacteriaceae, Acinetobacter OXA group: OXA-23, OXA-40, OXA-58, OXA-143 and the intrinsic OXA-51-like enzymes, OXA-198 in P. aeruginosa ). Materials and method The Amplidiag ® Carba-R + VRE, a qualitative multiplex nucleic acid-based in-vitro diagnostic test intended for the detection of carbapenemase-producing bacteria and vancomycin resistant enterococci, has been tested on a collection of 100 characterized Gram-negative isolates with reduced susceptibility to carbapenems, and on 200 isolates collected at the National Reference Center for Antibiotic Resistance from 20st January to 10th February 2016. The markers detected by this assay are bla KPC-lik e, bla NDM-like , bla VIM-like , bla IMP-like , bla OXA-48-like , Acinetobacter OXA genes including bla OXA-23 , bla OXA-24/-40 , bla OXA-58 , and bla OXA-51 including upstream promoter ISAba1 , and vanA and vanB . DNA was extracted using QiaAmp DNA extraction kits or by boiling extract. Results The Amplidiag ® CarbaR + VRE was able to detect all KPC, NDM, VIM, IMP, OXA-48 and variants including OXA−162, −181, −204, −232, −244. Similarly, all A. baumannii producing OXA−23, OXA−24/−40, OXA−58 as well as over expression of the chromosomally-encoded OXA−51-like b-lactamase, due to the presence of ISAba1 upstream of the blaOXA-51 gene were detected. The most prevalent carbapenemases encountered in P. aeruginosa have also been detected. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES−5 in P. aeruginosa , GES−14 in A. baumannii ), GIM−1, AIM−1, SPM−1, DIM−1 or OXA−198 in P. aeruginosa , or OXA−143-like in A. baumannii were not detected by the Amplidiag® CarbaR + VRE assay. The PCR worked equally well on purified DNA and on boiling extracted DNA from a colony. The biological performances of the Amplidiag® CarbaR + VRE on the prospective study of 200 CREs isolates collected at the NRC were of 100% and 96% sensitivity and specificity, respectively. Conclusion Amplidiag® Carba-R's performances were high as it was able to detect the five major carbapenemases: NDM, VIM, IMP, KPC, and OXA−48, as well as OXA-type carbapenemases from Acinetobacter sp. It can provide a result directly on colonies growing on selective screening media within two hours. This test needs now to be evaluated on rectal swabs, since it is able to detect the most worrisome resistant traits in Gram negative bacteria.