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ABSTRACTA novel mcrvariant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniaeisolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coliand K. pneumoniaestrains from geographically distant sites (Estonia, China, and South Africa).

ABSTRACTAn Enterobacter ludwigiistrain was isolated during routine screening of a Japanese patient for carriage of carbapenem-resistant Enterobacteriaceae. PCR analysis revealed the blaNMC-Acarbapenemase gene. Whole-genome sequencing revealed that blaNMC-Awas inserted in the chromosome and associated with a novel 29.1-kb putative Xer-dependent integrative mobile element, named EludIMEX-1. Bioinformatic analysis identified similar elements in the genomes of an Enterobacter asburiaestrain and of other Enterobacter cloacaecomplex strains, confirming the mobile nature of this element.

ABSTRACTThe In70.2 integron platform appears to be a conserved structure involved in the dissemination of the blaVIM-1metallo-β-lactamase gene in Pseudomonas aeruginosa. The genetic context of the In70.2 integron platform from P. aeruginosaVR-143/97, the VIM-1-producing index strain isolated in Italy in 1997, was fully characterized by a next-generation sequencing approach refined by conventional sequencing. The In70.2 integron platform from VR-143/97 was found to be associated with a defective Tn402-like transposon inserted into the urf2gene of a Tn3family transposon of an original structure, named Tn6249, which also carried a partially deleted meroperon and an In90 integron platform in a tail-to-tail orientation. Tn6249was inserted into a PACS171b-like genomic island, which was in turn inserted into the endAgene of the Pseudomonaschromosomal backbone. Tn6249showed a similar structure and a conserved location with respect to that of Tn6060, a Tn3family transposon associated with In70.2 and carrying a double-integron platform, which was detected in a VIM-1-producing P. aeruginosastrain isolated in Australia in 2008. Both Tn6249and Tn6060are apparently derived from Tn6162, a mercury resistance transposon carrying an integron platform, which was found in P. aeruginosaisolates from different geographic locations. The conservation of the genetic context of Tn6249and Tn6060suggests an in situevolution of these elements after the insertion of a Tn6162-like ancestor into the PACS171b-like genomic island (GI) present in the genome of a successful widespread P. aeruginosaclonal lineage.

ABSTRACTKlebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrBgene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrBgene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrBgene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrBlocus, while the remaining 27 strains (41%) did not show mgrBalterations. Transcriptional upregulation of the phoQand pmrKgenes (part of the phoPQand pmrHFIJKLMoperon, respectively) was observed in all strains with mgrBalterations. Complementation experiments with a wild-type mgrBgene restored colistin susceptibility and basal expression levels of phoQand pmrKgenes in strains carrying different types of mgrBalterations. The present results suggest that mgrBalteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

ABSTRACTColistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceaeproducing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrAand of pmrK, which is part of the pmrHFIJKLMoperon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrBin KPB-2 restored colistin susceptibility and reduced the transcription of pmrAand pmrKto basal levels, while expression of PmrBL82Rin KPB-1 did not alter colistin susceptibility or upregulate pmrAand pmrKexpression, confirming the dominance of wild-type PmrB versus the PmrBL82Rmutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

ABSTRACTColistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniaeproducing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrBgene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrBinactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrBdeletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrBinactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrBgene, upregulated transcription of phoP, phoQ, and pmrK(which is part of the pmrHFIJKLMoperon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniaeand were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLMoperon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniaestrains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401aKPC-3-encoding transposon.

ABSTRACTCarbapenem-resistant Klebsiella pneumoniaecauses important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae(OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB(distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.