Genomic Insights into Colistin-Resistant Klebsiella pneumoniae from a Tunisian Teaching Hospital

ABSTRACT The emergence of colistin-resistant Klebsiella pneumoniae (CoRKp) is a public health concern, since this antibiotic has become the last line of treatment for infections caused by multidrug-resistant (MDR) Gram negatives. In this study, we have investigated the molecular basis of colistin resistance in 13 MDR K. pneumoniae strains isolated from 12 patients in a teaching hospital in Sousse, Tunisia. Whole-genome sequencing (WGS) was used to decipher the molecular mechanism of colistin resistance and to identify the resistome of these CoRKp isolates. It revealed a genome of ca. 5.5 Mbp in size with a G+C content of 57%, corresponding to that commonly observed for K. pneumoniae. These isolates belonged to the 5 different sequence types (ST11, ST15, ST101, ST147, and ST392), and their resistome was composed of acquired β-lactamases, including extended-spectrum beta-lactamase and carbapenemase genes (bla CTX-M-15, bla OXA-204, bla OXA-48, and bla NDM-1 genes), aminoglycoside resistance genes [aac(6′)Ib-cr, aph(3″)-Ib, aph(6)-Id, and aac(3)-IIa], and fosfomycin (fosA), fluoroquinolone (qnr-like), chloramphenicol, trimethoprim, and tetracycline resistance genes. All of the isolates were identified as having a mutated mgrB gene. Mapping reads with reference sequences of the most common genes involved in colistin resistance revealed several modifications in mgrB, pmr, and pho operons (deletions, insertions, and substitutions) likely affecting the function of these proteins. It is worth noting that among the 12 patients, 10 were treated with colistin before the isolation of CoRKp. No plasmid encoding mcr-1 to mcr-5 genes was found in these isolates. This study corresponds to the first molecular characterization of a collection of CoRKp strains in Tunisia and highlights that the small-transmembrane protein MgrB is a main mechanism for colistin resistance in K. pneumoniae.

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Multihospital Occurrence of Pan-Resistant Klebsiella pneumoniae Sequence Type 147 with an ISEcp1-Directed blaOXA-181 Insertion in the mgrB Gene in the United Arab Emirates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00418-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 28

Author(s):

Ágnes Sonnevend ◽

Akela Ghazawi ◽

Rayhan Hashmey ◽

Aliasgher Haidermota ◽

Safinaz Girgis ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

South Korea ◽

United Arab Emirates ◽

Resistance Genes ◽

Extended Period ◽

Sequence Type ◽

Resistant Clone ◽

Colistin Resistance ◽

Content Type ◽

Resistance Island

ABSTRACT The emergence of pan-resistant Klebsiella pneumoniae strains is an increasing concern. In the present study, we describe a cluster of 9 pan-resistant K. pneumoniae sequence type 147 (ST147) isolates encountered in 4 patients over nearly 1 year in 3 hospitals of the United Arab Emirates (UAE). The isolates exhibited highly similar genotypes. All produced chromosomally encoded OXA-181, and the majority also produced the NDM-5 carbapenemase. As with the previously described single isolate from the UAE, MS6671, the mgrB was disrupted by a functional, ISEcp1-driven bla OXA-181 insertion causing resistance to carbapenems. The mutation was successfully complemented with an intact mgrB gene, indicating that it was responsible for colistin resistance. bla NDM-5 was located within a resistance island of an approximately 100-kb IncFII plasmid carrying ermB, mph(A), bla TEM-1B, rmtB, bla NDM-5, sul1, aadA2, and dfrA12 resistance genes. Sequencing this plasmid (pABC143-NDM) revealed that its backbone was nearly identical to that of plasmid pMS6671E from which several resistance genes, including bla NDM-5, had been deleted. More extensive similarities of the backbone and the resistance island were found between pABC143C-NDM and the bla NDM-5-carrying IncFII plasmids of two K. pneumoniae ST147 isolates from South Korea, one of which was colistin resistant, and both also produced OXA-181. Notably, one of these strains was isolated from a patient transferred from the UAE. Our data show that this pan-resistant clone has an alarming capacity to maintain itself over an extended period of time and is even likely to be transmitted internationally.

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Prevalence of Aminoglycoside Resistance Genes and Molecular Characterization of a Novel Gene, aac(3)-IIg, among Clinical Isolates of the Enterobacter cloacae Complex from a Chinese Teaching Hospital

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00852-20 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Xinyi Zhu ◽

Peizhen Li ◽

Changrui Qian ◽

Hongmao Liu ◽

Hailong Lin ◽

...

Keyword(s):

Teaching Hospital ◽

Resistance Genes ◽

High Throughput Sequencing ◽

Enterobacter Cloacae ◽

Human Pathogens ◽

Aminoglycoside Resistance ◽

Class 1 Integron ◽

Content Type ◽

Novel Gene ◽

Aminoglycoside Resistance Genes

ABSTRACT Members of the Enterobacter cloacae complex are important opportunistic human pathogens capable of causing a wide variety of infections. During recent decades, aminoglycoside-resistant E. cloacae complex isolates have increasingly been reported and have become a major concern. Here, we employed high-throughput sequencing in combination with specific PCR assays to investigate the prevalence of aminoglycoside resistance genes among 170 isolates of the E. cloacae complex collected from a teaching hospital in Wenzhou, China. A total of 12 known genes [aphA-1, strA, strB, aac(6′)-IIc, aadA2, aac(3)-IId, aadB, aadA1, rmtB, armA, aadA5, and aac(6′)-Ie–aph(2′')-Ia] and 1 novel gene [aac(3)-IIg] were identified, with aphA-1 (71.18%), strA (55.29%), and strB (52.35%) being the most prevalent, and aac(3)-IIg was detected with a positive rate of 21.76% (37/170). The aac(3)-IIg gene was 810 bp in length and encoded a protein that shared 72 to 78% identities with previously known AAC(3)-II aminoglycoside 3-N-acetyltransferases. The MICs of gentamicin and tobramycin were 512 μg/ml and 64 μg/ml, respectively, when aac(3)-IIg was cloned into Escherichia coli DH5α. All aac(3)-IIg-positive isolates exerted broad aminoglycoside resistance profiles, mediated by the coexistence of multiple resistance genes. Moreover, aminoglycoside resistance and resistance genes were found to be transferable in most strains (24/37). Nevertheless, pulsed-field gel electrophoresis (PFGE) and dendrogram analysis showed clonal diversity among these isolates. S1 nuclease PFGE, Southern hybridization, and whole-genome sequencing indicated that aac(3)-IIg was located on transferable as well as nontransferable plasmids of various sizes. The analysis of the genetic environment suggested that aac(3)-IIg is embedded within a class 1 integron, with IS26 playing an important role in its mobility.

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Colistin Resistance Gene mcr-8 in a High-Risk Sequence Type 15 Klebsiella pneumoniae Isolate from Kenya

Microbiology Resource Announcements ◽

10.1128/mra.00783-20 ◽

2020 ◽

Vol 9 (39) ◽

Author(s):

Cecilia Kyany’a ◽

Lillian Musila

Keyword(s):

High Risk ◽

Klebsiella Pneumoniae ◽

Resistance Gene ◽

Resistance Genes ◽

Multidrug Resistant ◽

Sequence Type ◽

Colistin Resistance ◽

Content Type ◽

Global Concern ◽

Hospitalized Patient

ABSTRACT The emergence and rise of mobile colistin resistance genes are of great global concern due to the ease of transfer of resistance to other bacteria. This report describes the genome of a colistin- and multidrug-resistant Klebsiella pneumoniae isolate bearing mcr-8, obtained from a hospitalized patient in Kenya.

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Reduced Fitness Costs of mcr-1.2 Compared to Mutated pmrB in Isogenic Colistin-Resistant KPC-3-Producing Klebsiella pneumoniae

mSphere ◽

10.1128/msphere.00551-19 ◽

2019 ◽

Vol 4 (6) ◽

Author(s):

Cesira Giordano ◽

Adrian Klak ◽

Simona Barnini ◽

Monika A. Chlebowicz ◽

Mariacristina Menconi ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Genomic Analysis ◽

Growth Curves ◽

Growth Characteristics ◽

Fitness Costs ◽

Aminoglycoside Resistance ◽

Content Type ◽

Pediatric Hematology ◽

Lag Phases

ABSTRACT In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage. IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2. An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.

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Genomic Characterization of Colistin Heteroresistance in Klebsiella pneumoniae during a Nosocomial Outbreak

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01344-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6837-6843 ◽

Cited By ~ 52

Author(s):

Teysir Halaby ◽

Emre Kucukkose ◽

Axel B. Janssen ◽

Malbert R. C. Rogers ◽

Dennis J. Doorduijn ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Population Analysis ◽

Resistant Bacteria ◽

Colistin Resistance ◽

Sequence Element ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

Genomic Characterization ◽

Insertion Sequence Element

ABSTRACTKlebsiella pneumoniaeis emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extended-spectrum-beta-lactamase (ESBL)-producingK. pneumoniaeisolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producingK. pneumoniaeisolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in theseK. pneumoniaeisolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in thelpxM,mgrB,phoQ, andyciMgenes in colistin-resistantK. pneumoniaeisolates. In two strains,mgrBwas inactivated by an IS3-like or ISKpn14insertion sequence element. Complementation intranswith the wild-typemgrBgene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2- to 4-fold in the presence of the mutatedphoQ,lpxM, andyciMalleles. In conclusion, the present study indicates that heteroresistantK. pneumoniaesubpopulations may be selected for upon exposure to colistin. Mutations inmgrBandphoQhave previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in theyciMandlpxMgenes in the emergence of colistin resistance inK. pneumoniae.

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Colistin Resistance in Carbapenem-Resistant Klebsiella pneumoniae Mediated by Chromosomal Integration of Plasmid DNA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00404-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 2

Author(s):

Astrid V. Cienfuegos-Gallet ◽

Liang Chen ◽

Barry N. Kreiswirth ◽

J. Natalia Jiménez

Keyword(s):

Klebsiella Pneumoniae ◽

Plasmid Dna ◽

Clonal Expansion ◽

Genetic Alterations ◽

Sequence Type ◽

Chromosomal Integration ◽

Colistin Resistance ◽

Content Type ◽

Carbapenem Resistant ◽

Single Locus Variant

ABSTRACT Here we describe the spread of colistin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae in Medellín, Colombia. Among 32 isolates collected between 2012 and 2014, 24 showed genetic alterations in mgrB. Nineteen isolates belonged to sequence type 512 (ST512) (or its single locus variant [SLV]) and harbored an 8.1-kb hsdMSR insertion corresponding to ISKpn25, indicating a clonal expansion of the resistant strain. The insertion region showed 100% identity to several plasmids, suggesting that the colistin resistance is mediated by chromosomal integration of plasmid DNA.

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Klebsiella pneumoniae ST258 Producing KPC-3 Identified in Italy Carries Novel Plasmids and OmpK36/OmpK35 Porin Variants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05308-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 2143-2145 ◽

Cited By ~ 107

Author(s):

Aurora García-Fernández ◽

Laura Villa ◽

Claudio Carta ◽

Carolina Venditti ◽

Alessandra Giordano ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Uptake System ◽

Antimicrobial Drugs ◽

Toxic Compounds ◽

Content Type ◽

Plasmid Content

ABSTRACTA carbapenemase-resistantKlebsiella pneumoniaestrain, clone ST258 producing KPC-3, was fully characterized. The entire plasmid content was investigated, thereby identifying plasmids of the IncFIIk(two of them similar to pKPQIL and pKPN3, respectively), IncX, and ColE types, carrying a formidable set of resistance genes against toxic compounds, metals, and antimicrobial drugs and a novel iron(III) uptake system.

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Comparison of the Superpolymyxin and ChromID Colistin R Screening Media for the Detection of Colistin-Resistant Enterobacteriaceae from Spiked Rectal Swabs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01618-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 7

Author(s):

Delphine Girlich ◽

Thierry Naas ◽

Laurent Dortet

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Confidence Interval ◽

Enterobacter Cloacae ◽

Bacterial Isolates ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Rectal Swabs ◽

Stool Samples

ABSTRACT The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has led to the increased use of colistin, which has resulted in the emergence of colistin-resistant Enterobacteriaceae worldwide. One of the most threatening scenarios is the dissemination of colistin resistance in CPE, particularly the plasmid-encoded resistance element MCR. Thus, it has now become mandatory to possess reliable media to screen for colistin-resistant Gram-negative bacterial isolates, especially Enterobacteriaceae. In this study, we evaluated the performances of the Superpolymyxin medium (ELITechGroup) and the ChromID Colistin R medium (bioMérieux) to screen for colistin-resistant Enterobacteriaceae from spiked rectal swabs. Stool samples were spiked with a total of 94 enterobacterial isolates (Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Enterobacter cloacae), including 53 colistin-resistant isolates. ESwabs (Copan Diagnostics) were then inoculated with those spiked fecal suspensions, and culture proceeded as recommended by both manufacturers. The sensitivity of detection of colistin-resistant Enterobacteriaceae was 86.8% (95% confidence interval [95% CI] = 74.0% to 94.0%) using both the Superpolymyxin medium and the ChromID Colistin R plates. Surprisingly, the isolates that were not detected were not the same for both media. The specificities were high for both media, at 97.9% (95% CI = 87.3% to 99.9%) for the Superpolymyxin medium and 100% (95% CI = 90.4% to 100%) for the ChromID Colistin R medium. Both commercially available media, ChromID Colistin R and Superpolymyxin, provide useful tools to screen for colistin-resistant Enterobacteriaceae from patient samples (rectal swabs) regardless of the level and mechanism of colistin resistance.

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High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids

Frontiers in Microbiology ◽

10.3389/fmicb.2021.636396 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xueya Zhang ◽

Qiaoling Li ◽

Hailong Lin ◽

Wangxiao Zhou ◽

Changrui Qian ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Genomic Analysis ◽

Comparative Genomic Analysis ◽

Conjugative Plasmid ◽

Comparative Genomic ◽

Aminoglycoside Resistance ◽

Life Threatening ◽

Aminoglycoside Resistance Genes ◽

High Level

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA-carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA-carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination.

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Coexistence of aminoglycoside resistance genes in CTX-M-producing isolates of Klebsiella pneumoniae in Bushehr province, Iran

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i2.5975 ◽

2021 ◽

Author(s):

Behrouz Latifi ◽

Saeed Tajbakhsh ◽

Leila Ahadi ◽

Forough Yousefi

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Confirmatory Test ◽

M Gene ◽

Aminoglycoside Resistance ◽

Genetic Groups ◽

Genes Encoding ◽

Resistance Patterns ◽

Aminoglycoside Resistance Genes ◽

Antibiotic Resistance Patterns

Background and Objectives: Increasing the rate of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae has given rise to a major healthcare issue in clinical settings over the past few years. Treatment of these strains is hardly effective since the plasmid encoding ESBL may also carry other resistance genes including aminoglycosides. The current study aimed to evaluate the prevalence of ESBL-producing K. pneumoniae and investigate the coexistence of Cefoxitamase-Munich (bla ) with aminoglycoside-modifying enzyme (AME) genes, aac(3)IIa as well as aac(6′)Ib, in CTX‑M‑producing K. pneumoniae isolated from patients in Bushehr province, Iran. Materials and Methods: A total of 212 K. pneumoniae isolates were collected and confirmed using polymerase chain re‑ action (PCR) of the malate dehydrogenase gene. Isolates were screened for production of ESBL. Phenotypic confirmatory test was performed using combined disk test. The genes encoding CTX-M groups and AME genes, aac(3)IIa and aac(6′)Ib, were investigated by PCR. Results: The ESBL phenotype was detected in 56 (26.4%) K. pneumoniae isolates. Moreover, 83.9% of ESBL-producing isolates carried the genes for CTX-M type β-lactamases, which were distributed into the two genetic groups of CTX-M-1 (97.8%)- and CTX-M-2 (2.1%)-related enzymes. Notably, among K. pneumoniae isolates containing the blaCTX‑M gene, 68.08% of isolates harbored AME genes. In addition, the coexistence of bla in 46.8% of CTX-M-producing K. pneumoniae isolates. Conclusion: This study provides evidence of a high prevalence of AME genes in CTX-M- producing K. pneumoniae iso‑ lates; therefore, in the initial empirical treatment of infections caused by ESBL-KP in regions with such antibiotic resistance patterns, aminoglycoside combination therapy should be undertaken carefully.

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