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Multidrug Resistant Klebsiella pneumoniae ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

ABSTRACT: In this paper we describe the transmission of a multi-drug resistant Klebsiella pneumoniae ST101 clone from hospital to wastewater and its persistence after chlorine treatment. Water samples from influents and effluents of the sewage tank of an infectious diseases hospital and clinical strains collected from the intra-hospital infections, during a period of 10 days prior to wastewater sampling were analyzed. Antibiotic resistant K. pneumoniae strains from wastewaters were recovered on selective media. Based on antibiotic susceptibility profiles and PCR analyses of antibiotic resistance (AR) genetic background, as well as whole-genome sequencing (Illumina MiSeq) and subsequent bioinformatic analyses, 11 ST101 K. pneumoniae strains isolated from hospital wastewater influent, wastewater effluent and clinical sector were identified as clonally related. The SNP and core genome analyses pointed out that five strains were found to be closely related (with ?18 SNPs and identical cgMLST profile). The strains belonging to this clone harbored multiple acquired AR genes [bla _CTX-M- ₁₅, bla _OXA- ₄₈, bla _OXA- ₁, bla _SHV- ₁₀₆, bla _TEM- ₁₅₀, aac(3)-IIa, aac(6')-Ib-cr, oqxA10, oqxB17, fosA, catB3, dfrA14, tet(D)] and chromosomal mutations involved in AR (?mgrB, ?ompK35, amino acid substitutions in GyrA Ser83Tyr, Asp87Asn, ParC Ser80Tyr). Twenty-nine virulence genes involved in iron acquisition, biofilm and pili formation, adherence, and the type six secretion system - T6SS-III were identified. Our study proves the transmission of MDR K. pneumoniae from hospital to the hospital effluent and its persistence after the chlorine treatment, raising the risk of surface water contamination and further dissemination to different components of the trophic chain, including humans.

SUBMITTER: Popa LI

PROVIDER: S-EPMC7873994 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Multidrug Resistant Klebsiella pneumoniae ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

Popa Laura Ioana LI Gheorghe Irina I Barbu Ilda Czobor IC Surleac Marius M Paraschiv Simona S Măruţescu Luminiţa L Popa Marcela M Pîrcălăbioru Graţiela Grădişteanu GG Talapan Daniela D Niţă Mihai M Streinu-Cercel Anca A Streinu-Cercel Adrian A Oţelea Dan D Chifiriuc Mariana Carmen MC

Frontiers in microbiology 20210111

In this paper we describe the transmission of a multi-drug resistant Klebsiella pneumoniae ST101 clone from hospital to wastewater and its persistence after chlorine treatment. Water samples from influents and effluents of the sewage tank of an infectious diseases hospital and clinical strains collected from the intra-hospital infections, during a period of 10 days prior to wastewater sampling were analyzed. Antibiotic resistant K. pneumoniae strains from wastewaters were recovered ...[more]

PMID: 33584574

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Project description:Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Project description:Five blaCTX-M-14-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.

Project description:BackgroundKlebsiella pneumoniae ST101 is an emerging high-risk clone which exhibits extensive drug resistance. Bacterial strains residing in multiple hosts show unique signatures related to host adaptation. In this study, we assess the genetic relationship of K. pneumoniae ST101 isolated from hospital samples, the environment, community, and livestock using whole genome sequencing (WGS).Materials and methodsWe selected ten K. pneumoniae ST101 strains from hospitalized patients in Italy (n = 3) (2014) and Spain (n = 5) (2015-2016) as well as Belgian livestock animals (n = 2) (2017-2018). WGS was performed with 2 × 250 bp paired-end sequencing (Nextera XT) sample preparation kit and MiSeq (Illumina Inc.). Long-read sequencing (Pacbio Sequel I) was used to sequence the two livestock strains and three Italian hospital-associated strains. Furthermore, a public ST101 sequence collection of 586 strains (566 hospital-associated strains, 12 environmental strains, six strains from healthy individuals, one food-associated strain and one pig strain) was obtained. BacPipe and Kleborate were used to conduct genome analysis. ISFinder was used to find IS elements, and PHASTER was utilized to identify prophages. A phylogenetic tree was constructed to illustrate genetic relatedness.ResultsHospital-associated K. pneumoniae ST101 showed higher resistance scores than non-clinical isolates from healthy individuals, the environment, food and livestock (1.85 ± 0.72 in hospital-associated isolates vs. 1.14 ± 1.13 in non-clinical isolates, p < 0.01). Importantly, the lack of integrative conjugative elements ICEKp bearing iron-scavenging yersiniabactin siderophores (ybt) in livestock-associated strains suggests a lower pathogenicity potential than hospital-associated strains. Mobile genetic elements (MGE) appear to be an important source of diversity in K. pneumoniae ST101 strains from different origins, with a highly stable genome and few recombination events outside the prophage-containing regions. Core genome MLST based analysis revealed a distinct genetic clustering between human and livestock-associated isolates.ConclusionThe study of K. pneumoniae ST101 hospital-associated and strains from healthy individuals and animals revealed a genetic diversity between these two groups, allowing us to identify the presence of yersiniabactin siderophores in hospital-associated isolates. Resistance and virulence levels in livestock-associated strains were considerably lower than hospital-associated strains, implying that the public health risk remains low. The introduction of an ICEKp into animal strains, on the other hand, might pose a public threat over time.

Project description:Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of bla KPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A bla KPC-2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

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Multidrug Resistant Klebsiella pneumoniae ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

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Multidrug Resistant <i>Klebsiella pneumoniae</i> ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

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