Performance of two methods of carbapenem-resistant Enterobacterales surveillance on a kidney transplant ward: selective culture of and real-time PCR directly from rectal swabs.

BACKGROUND: Infection with carbapenem-resistant Enterobacterales (CRE) is associated with a high mortality rate in kidney transplant recipients, and colonization with CRE is one of the major risk factors for CRE infection. There is, therefore, a need to improve the capacity to detect colonization with CRE among inpatients. METHODS: In this prospective study, we compared the performance of real-time PCR for carbapenemase directly from rectal swabs with that of conventional CRE surveillance culture in all patients admitted to a kidney transplant ward between February 2019 and March 2020. Surveillance culture and real-time PCR were performed at admission and weekly until hospital discharge. Two perineum-rectal swabs were collected: one for culture and one for PCR. RESULTS: We collected 905 paired samples for CRE surveillance from 399 patients, of whom 347 (87.0%) were kidney transplant recipients and 52 were waiting list patients. CRE was detected by culture and/or PCR in 75 patients (18.8%). Positivity for CRE was identified by PCR in 62 (15.5%) of the 399 patients and by culture in 55 (13.8%); 20 (5.0%) of the patients tested positive only on PCR, and 13 (3.3%) tested positive only on culture. The most common carbapenemase and species were, respectively, blaKPC (in 85.5%) and Klebsiella pneumoniae (in 80.0%). Infection with CRE occurred in 21.6% of the colonized patients, those cases occurred only among kidney transplant recipients. None of the patients who tested negative on culture developed CRE infection. CONCLUSION: In conclusion, the two methods are complementary and could be useful in a scenario of high CRE prevalence.

Linear plasmids in Klebsiella and other Enterobacteriaceae.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella, first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K. pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Extensively drug-resistant IMP-16-producing Pseudomonas monteilii isolated from cerebrospinal fluid.

IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and since then, bacteria with this metallo-ß-lactamase have been detected worldwide. Pseudomonas monteilii (part of P. putida group) were considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged. The present study reports the draft sequence of an extensively drug-resistant IMP-16-producing P. monteilii 597/14 isolated from cerebrospinal fluid in 2014. The sequencing data revealed blaIMP-16 as a gene cassette on class 1 integron, In1738 characterized in this study. Furthermore, the resistome of Pm597/14 consisted of 7 resistance genes (aadA1b, strA, strB, aacA4, blaIMP-16, blaOXA-2, sul1) and diverse virulence determinants involved in the adherence, LPS, antiphagocytosis, iron uptake and mercuric resistance. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection. P. monteilii isolates have been reported from clinical and environmental sources, carrying different MBL genes showing its potential role as their reservoir.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

OBJECTIVES: Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. METHODS: ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. RESULTS: Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to ß-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. CONCLUSION: This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

The role of therapy with aminoglycoside in the outcomes of kidney transplant recipients infected with polymyxin- and carbapenem-resistant Enterobacteriaceae.

Kidney transplant recipients are at risk for infections due to carbapenem-resistant Enterobacteriaceae (CRE). Polymyxin-resistant CRE (PR-CRE) infections are especially difficult to treat. The aim of this study was to characterize PR-CRE infections among kidney transplant recipients and identify risk factors for treatment failure. This retrospective cohort study involved all kidney transplant recipients with PR-CRE infection between 2013 and 2017 at our center. Minimal inhibitory concentrations for polymyxin B were determined by broth microdilution. Carbapenem-resistant genes (blaKPC, blaNDM, and blaOXA-48), aminoglycoside-resistance genes, and polymyxin-resistant gene mcr-1 were identified by polymerase chain reaction. All but one of the 47PR-CRE infections identified were due to Klebsiella pneumoniae. The most common type of infection (in 54.3%) was urinary tract infection (UTI). Monotherapy was used in 10 cases. Combined treatment regimens included double-carbapenem therapy in 19 cases, oral fosfomycin in 19, and amikacin in 13. Treatment failure occurred in 21 cases (45.7%). Clinical success was achieved 78.9% of patients who used aminoglycosides versus 37.0% of those who not used this drug (p = 0.007). Multivariate analysis showed diabetes mellitus to be a risk factor for treatment failure; amikacin use and UTI were found to be protective. Nine strains were RmtB producers. Although aminoglycosides constitute an important therapeutic option for PR-CRE infection, the emergence of aminoglycoside resistance could have a major impact on the management of CRE infection.

Draft genome sequences of KPC-2- and CTX-M-15-producing Klebsiella pneumoniae ST437 isolated from a clinical sample and urban rivers in Sao Paulo, Brazil.

OBJECTIVES: KPC-producing Klebsiella pneumoniae is considered one of the most worrisome multidrug-resistant micro-organisms in nosocomial infections. It has also been reported in wastewater and urban rivers in the city of Sao Paulo, Brazil. Here we report the draft genome sequences of three KPC-2- and CTX-M-15-producing K. pneumoniae sequence type 437 (ST437) isolates obtained from two urban rivers and from a clinical sample of a patient in Sao Paulo. METHODS: A genomic library was constructed using a Nextera XT Kit. An Illumina platform was used to perform whole-genome sequencing (WGS). RESULTS: WGS of environmental isolates Kp148/PINH-4900 and Kp196/TIET-4200 and clinical isolate Kp314/11 resulted in estimated genome sizes of 5464058, 5437723 and 5319218bp, respectively. Resistome analysis of the environmental and clinical strains revealed the presence of resistance genes to the following antimicrobials in all strains: aminoglycosides [aac(6')-Ib-cr]; ß-lactams (blaOXA-1, blaSHV-11, blaCTX-M-15 and blaKPC-2); fluoroquinolones [aac(6')-Ib-cr, oqxA and oqxB]; fosfomycin (fosAKP); macrolides [mph(A)]; phenicols (catB4); sulfonamides (sul1); and trimethoprim (dfrA30). The tetracycline resistance gene tetA was identified in Kp148/PINH-4900 and Kp314/11 only; the aminoglycoside resistance gene aph(3')-Ia was found only in environmental isolates, and aadA2 only in Kp314/11; and the phenicol resistance gene catA1 was identified only in Kp148/PINH-4900. CONCLUSIONS: The draft genome sequences of these strains help us to elucidate the dissemination of resistance genes in micro-organisms inside and outside the hospital and are useful for further comparisons between clinical and environmental strains.

Endemicity of the High-Risk Clone Klebsiella pneumoniae ST340 Coproducing QnrB, CTX-M-15, and KPC-2 in a Brazilian Hospital.

The dissemination of multiresistant Klebsiella pneumoniae carbapenemase (KPC)-2-producing Klebsiella pneumoniae isolates belonging to international high-risk clones poses a major health care threat. In this study, 48 nonduplicated, carbapenem-resistant K. pneumoniae isolated from 2011 to 2014 in a tertiary hospital were investigated. The blaKPC-2 gene was the only determinant for carbapenem resistance. The blaCTX-M-15 gene was the main determinant for the production of extended-spectrum beta-lactamase (ESBL), whereas aph(3')-Ia and qnrB were the most common genes associated with resistance to aminoglycosides and quinolones, respectively. Nine different sequence types (STs) were identified. The most common was ST340. Molecular typing by enterobacterial repetitive intergenic consensus-PCR placed 48 strains among 10 different clusters. In the studied hospital, the high-risk clone of KPC-2-producing K. pneumoniae ST340, harboring genes that codify aminoglycoside modifying enzymes, QnrB and CTX-M-15 plus CTXM-2-type ESBLs, is disseminated and acts as a major agent of infections in critically ill patients.

Draft genome sequence of a KPC-2-producing Klebsiella pneumoniae ST340 carrying blaCTX-M-15 and blaCTX-M-59 genes: a rich genome of mobile genetic elements and genes encoding antibiotic resistance.

OBJECTIVES: Klebsiella pneumoniae is considered an opportunistic pathogen and an important agent of nosocomial and community infections. It presents the ability to capture and harbour several antimicrobial resistance genes and, in this context, the extensive use of carbapenems to treat serious infections has been responsible for the selection of several resistance genes. This study reports the draft genome sequence of a KPC-2-producing K. pneumoniae strain (Kp10) simultaneously harbouring blaCTX-M-15 and blaCTX-M-59 genes isolated from urine culture of a patient with Parkinson's disease. METHODS: Classical microbiological methods were applied to isolate and identify the strain, and PCR and sequencing were used to identify and characterise the genes and the genetic environment. Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and a NextSeq platform. RESULTS: WGS analysis revealed the presence of 5915 coding genes, 46 RNA-encoding genes and 255 pseudogenes. Kp10 belonged to sequence type 340 (ST340) of clonal complex 258 (CC258) and carried 20 transferable genes associated with antimicrobial resistance, comprising seven drug classes. Although the simultaneous presence of different blaCTX-M genes in the same strain is rarely reported, the blaKPC-2, blaCTX-M-15 and blaCTX-M-59 genes were not associated with the same genetic mobile structure in Kp10. CONCLUSIONS: These results confirm the capacity of K. pneumoniae to harbour several antimicrobial resistance genes. Thus, this draft genome could help in future epidemiological studies regarding the dissemination of clinically relevant resistance genes.

IncX3 plasmid harboring a non-Tn4401 genetic element (NTEKPC) in a hospital-associated clone of KPC-2-producing Klebsiella pneumoniae ST340/CG258.

IncX-type plasmids have achieved clinical significance for their contribution in the dissemination of genes confering resistance to carbapenems (most blaKPC- and blaNDM-type genes) and polymyxins (mcr-type genes), both antibiotics considered last resort for multidrug-resistant Gram-negative infections. In this study, we report the identification and complete sequence analysis of an IncX3 plasmid (designated pKP1194a) carrying a non-Tn4401 genetic element (NTEKPC) of tnpR-tnpA (partial)-blaKPC-2-ΔISKpn6/traN, originating from a hospital-associated lineage of K. pneumoniae belonging to the ST340/CG258, with epidemiological link to Brazil.

Transfer of KPC-2 carbapenemase from Klebsiella pneumoniae to Enterobacter cloacae in a patient receiving meropenem therapy.

The horizontal transfer of a plasmid bearing the blaKPC-2 gene from K. pneumoniae to E. cloacae infecting the respiratory tract of a patient during meropenem therapy was elucidated. This finding is particularly worrisome, since these drugs are of last resort for multidrug-resistant Gram-negative pathogens.

Draft genome sequence of an aminoglycoside-resistant RmtG-producing Pseudomonas aeruginosa ST235 isolated from a cystic fibrosis patient.

Cystic fibrosis (CF) patients are often chronically colonised or infected by non-fermenting Gram-negative bacilli, with Pseudomonas aeruginosa being the most prevalent. In this study, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain belonging to sequence type ST235, isolated from the respiratory tract of a CF patient with chronic colonisation. Whole-genome sequencing analysis revealed a 6.7Mb genome size and the presence of 12 antibiotic resistance genes, including the rmtG gene conferring high-level aminoglycoside resistance, located on the chromosome.

Draft Genome Sequence of a Hospital-Associated Clone of Klebsiella pneumoniae ST340/CC258 Coproducing RmtG and KPC-2 Isolated from a Pediatric Patient.

We report here the draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to the hospital-associated sequence type 340 (ST340; clonal complex CC258), isolated from a catheter tip culture from a pediatric patient. The multidrug-resistant strain coproduced the 16S rRNA methyltransferase rRNA RmtG and ß-lactamases KPC-2 and CTX-M-15.

Draft genome sequence of a CTX-M-15-producing Klebsiella pneumoniae sequence type 340 (clonal complex 258) isolate from a food-producing animal.

Klebsiella pneumoniae carrying blaCTX-M-15 have been widely disseminated in hospital settings. In this regard, most clinically important strains belong to clonal complex 28 (CC258), which includes sequence type 340 (ST340). In this study, we present the draft genome sequence of a CTX-M-15-producing ST340 K. pneumoniae strain isolated from a food-producing animal in Brazil.

Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene.

During a Brazilian multicentric antimicrobial resistance surveillance study, colistin resistance was investigated in 4,620 Enterobacteriaceae isolated from human, animal, food and environmental samples collected from 2000 to 2016. We present evidence that mcr-1-positive Escherichia coli has been emerging in South America since at least 2012, supporting a previous report on the possible acquisition of mcr-1-harbouring E. coli by European travellers visiting Latin American countries.

Outbreak of IMP-producing carbapenem-resistant Enterobacter gergoviae among kidney transplant recipients.

OBJECTIVES: The objective of this study was to investigate a prolonged outbreak of carbapenem-resistant Enterobacter gergoviae (CREG) involving kidney transplant recipients (KTRs) between 2009 and 2014. METHODS: A case-control study was undertaken. Controls (n = 52) were selected from CREG-negative KTRs. Surveillance cultures for CREG were collected weekly. Colonization was defined as isolation of CREG from surveillance samples or from clinical specimens, with no evidence of infection. We also investigated infection control practices at the facility. RESULTS: Of 26 identified cases, 13 had had no known contact with another CREG-positive patient before the first positive culture. Seven patients (27%) developed infection. The site most often colonized was the urinary tract. During the study period two clusters were identified, one in 2009 and another in 2013-14. DNA sequencing revealed blaIMP-1 in all CREG tested. No environmental or hand cultures tested positive for CREG. An audit of infection control practices detected flaws in the handling and cleaning of urinary tract devices. Multivariate analysis identified advanced age, ureteral stent use, retransplantation and male gender as risk factors for CREG acquisition. CONCLUSIONS: An outbreak among KTRs caused by an unusual species of MDR bacteria may have resulted from a common source of contamination related to urinary tract devices.

Complete Sequences of Multidrug Resistance Plasmids Bearing rmtD1 and rmtD2 16S rRNA Methyltransferase Genes.

Complete nucleotide sequences were determined for two plasmids bearing rmtD group 16S rRNA methyltransferase genes. pKp64/11 was 78 kb in size, belonged to the IncL/M group, and harbored blaTEM-1b, sul1, qacEΔ1, dfrA22, and rmtD1 across two multidrug resistance regions (MRRs). pKp368/10 was 170 kb in size, belonged to the IncA/C group, and harbored acrB, sul1, qacEΔ1, ant(3″)-Ia, aac(6')-Ib, cat, rmtD2, and blaCTX-M-8 across three MRRs. The rmtD-containing regions shared a conserved motif, suggesting a common origin for the two rmtD alleles.

Coproduction of 16S rRNA methyltransferase RmtD or RmtG with KPC-2 and CTX-M group extended-spectrum ß-lactamases in Klebsiella pneumoniae.

Eight Klebsiella pneumoniae clinical strains with high-level aminoglycoside resistance were collected from eight hospitals in São Paulo State, Brazil, in 2010 and 2011. Three of them produced an RmtD group 16S rRNA methyltransferase, RmtD1 or RmtD2. Five strains were found to produce a novel 16S rRNA methyltransferase, designated RmtG, which shared 57 to 58% amino acid identity with RmtD1 and RmtD2. Seven strains coproduced KPC-2 with or without various CTX-M group extended-spectrum ß-lactamases, while the remaining strain coproduced CTX-M-2.

Multiclonal outbreak of Klebsiella pneumoniae producing extended-spectrum beta-lactamase CTX-M-2 and novel variant CTX-M-59 in a neonatal intensive care unit in Brazil.

An outbreak of cephalosporin-resistant Klebsiella pneumoniae occurred in a neonatal intensive care unit in São Paulo, Brazil. Of the 10 pulsotypes identified during the outbreak and follow-up periods, nine produced CTX-M-2 or its new variant CTX-M-59 and one produced SHV-5. bla(CTX-M-2/59) genes were located on closely related plasmids that were transferable.