Multiplex PCR Analysis for Rapid Detection of Klebsiella pneumoniae Carbapenem-Resistant (Sequence Type 258 [ST258] and ST11) and Hypervirulent (ST23, ST65, ST86, and ST375) Strains

ABSTRACT Carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains have emerged recently. These strains are both hypervirulent and multidrug resistant and may also be highly transmissible and able to cause severe infections in both the hospital and the community. Clinical and public health needs require a rapid and comprehensive molecular detection assay to identify and track the spread of these strains and provide timely infection control information. Here, we develop a rapid multiplex PCR assay capable of distinguishing K. pneumoniae carbapenem-resistant isolates of sequence type 258 (ST258) and ST11, and hypervirulent ST23, ST65/ST375, and ST86 clones, as well as capsular types K1, K2, K locus type 47 (KL47), and KL64, and virulence genes rmpA, rmpA2, iutA, and iroN. The assay demonstrated 100% concordance with 118 previously genotyped K. pneumoniae isolates and revealed different populations of carbapenem-resistant and hypervirulent strains in two collections in China and the United States. The results showed that carbapenem-resistant and hypervirulent K. pneumoniae strains are still rare in the United States, whereas in China, ∼50% of carbapenem-resistant strains carry rmpA/rmpA2 and iutA virulence genes, which are largely associated with the epidemic ST11 strains. Similarly, a high prevalence of hypervirulent strains was found in carbapenem-susceptible isolates in two Chinese hospitals, but these primarily belong to ST23, ST65/ST375, and ST86, which are distinct from the carbapenem-resistant strains. Taken together, our results demonstrated that this PCR assay can be a useful tool for molecular surveillance of carbapenem-resistant and hypervirulent K. pneumoniae strains.

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Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States

mBio ◽

10.1128/mbio.02881-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Victor I. Band ◽

Sarah W. Satola ◽

Richard D. Smith ◽

David A. Hufnagel ◽

Chris Bower ◽

...

Keyword(s):

United States ◽

Klebsiella Pneumoniae ◽

Diagnostic Testing ◽

Cladistic Analysis ◽

The United States ◽

Clinical Diagnostics ◽

Infection Model ◽

Content Type ◽

Carbapenem Resistant ◽

Clinical Diagnostic

ABSTRACT Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States. IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

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Vaccine Protection against Multidrug-Resistant Klebsiella pneumoniae in a Nonhuman Primate Model of Severe Lower Respiratory Tract Infection

mBio ◽

10.1128/mbio.02994-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 3

Author(s):

Natalia Malachowa ◽

Scott D. Kobayashi ◽

Adeline R. Porter ◽

Brett Freedman ◽

Patrick W. Hanley ◽

...

Keyword(s):

United States ◽

Risk Factors ◽

Health Care ◽

Lower Respiratory Tract ◽

The United States ◽

Multidrug Resistant ◽

Sequence Type ◽

Content Type ◽

Carbapenem Resistant ◽

Cynomolgus Macaques

ABSTRACT Klebsiella pneumoniae is a human gut communal organism and notorious opportunistic pathogen. The relative high burden of asymptomatic colonization by K. pneumoniae is often compounded by multidrug resistance—a potential problem for individuals with significant comorbidities or other risk factors for infection. A carbapenem-resistant K. pneumoniae strain classified as multilocus sequence type 258 (ST258) is widespread in the United States and is usually multidrug resistant. Thus, treatment of ST258 infections is often difficult. Inasmuch as new preventive and/or therapeutic measures are needed for treatment of such infections, we developed an ST258 pneumonia model in cynomolgus macaques and tested the ability of an ST258 capsule polysaccharide type 2 (CPS2) vaccine to moderate disease severity. Compared with sham-vaccinated animals, those vaccinated with ST258 CPS2 had significantly less disease as assessed by radiography 24 h after intrabronchial installation of 108 CFU of ST258. All macaques vaccinated with CPS2 ultimately developed ST258-specific antibodies that significantly enhanced serum bactericidal activity and killing of ST258 by macaque neutrophils ex vivo. Consistent with a protective immune response to CPS2, transcripts encoding inflammatory mediators were increased in infected lung tissues obtained from CPS-vaccinated animals compared with control, sham-vaccinated macaques. Taken together, our data provide support for the idea that vaccination with ST258 CPS can be used to prevent or moderate infections caused by ST258. As with studies performed decades earlier, we propose that this prime-boost vaccination approach can be extended to include multiple capsule types. IMPORTANCE Multidrug-resistant bacteria continue to be a major problem worldwide, especially among individuals with significant comorbidities and other risk factors for infection. K. pneumoniae is among the leading causes of health care-associated infections, and the organism is often resistant to multiple classes of antibiotics. A carbapenem-resistant K. pneumoniae strain known as multilocus sequence type 258 (ST258) is the predominant carbapenem-resistant Enterobacteriaceae in the health care setting in the United States. Infections caused by ST258 are often difficult to treat and new prophylactic measures and therapeutic approaches are needed. To that end, we developed a lower respiratory tract infection model in cynomolgus macaques in which to test the ability of ST258 CPS to protect against severe ST258 infection.

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Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli, Including Sequence Type 131 (ST131), from Retail Chicken Breasts in the United States in 2013

Applied and Environmental Microbiology ◽

10.1128/aem.02956-16 ◽

2017 ◽

Vol 83 (6) ◽

Cited By ~ 15

Author(s):

James R. Johnson ◽

Stephen B. Porter ◽

Brian Johnston ◽

Paul Thuras ◽

Sarah Clock ◽

...

Keyword(s):

United States ◽

Escherichia Coli ◽

Virulence Genes ◽

Meat Products ◽

The United States ◽

Sequence Type ◽

The Other ◽

Antibiotic Resistant ◽

Content Type ◽

E Coli

ABSTRACT Chicken meat products are hypothesized to be vehicles for transmitting antimicrobial-resistant and extraintestinal pathogenic Escherichia coli (ExPEC) to consumers. To reassess this hypothesis in the current era of heightened concerns about antimicrobial use in food animals, we analyzed 175 chicken-source E. coli isolates from a 2013 Consumer Reports national survey. Isolates were screened by PCR for ExPEC-defining virulence genes. The 25 ExPEC isolates (12% of 175) and a 2:1 randomly selected set of 50 non-ExPEC isolates were assessed for their phylogenetic/clonal backgrounds and virulence genotypes for comparison with their resistance profiles and the claims on the retail packaging label (“organic,” “no antibiotics,” and “natural”). Compared with the findings for non-ExPEC isolates, the group of ExPEC isolates had a higher prevalence of phylogroup B2 isolates (44% versus 4%; P < 0.001) and a lower prevalence of phylogroup A isolates (4% versus 30%; P = 0.001), a higher prevalence of multiple individual virulence genes, higher virulence scores (median, 11 [range, 4 to 16] versus 8 [range, 1 to 14]; P = 0.001), and higher resistance scores (median, 4 [range, 0 to 8] versus 3 [range, 0 to 10]; P < 0.001). All five isolates of sequence type 131 (ST131) were ExPEC (P = 0.003), were as extensively resistant as the other isolates tested, and had higher virulence scores than the other isolates tested (median, 12 [range, 11 to 13] versus 8 [range, 1 to 16]; P = 0.005). Organic labeling predicted lower resistance scores (median, 2 [range, 0 to 3] versus 4 [range, 0 to 10]; P = 0.008) but no difference in ExPEC status or virulence scores. These findings document a persisting reservoir of extensively antimicrobial-resistant ExPEC isolates, including isolates from ST131, in retail chicken products in the United States, suggesting a potential public health threat. IMPORTANCE We found that among Escherichia coli isolates from retail chicken meat products purchased across the United States in 2013 (many of these isolates being extensively antibiotic resistant), a minority had genetic profiles suggesting an ability to cause extraintestinal infections in humans, such as urinary tract infection, implying a risk of foodborne disease. Although isolates from products labeled “organic” were less extensively antibiotic resistant than other isolates, they did not appear to be less virulent. These findings suggest that retail chicken products in the United States, even if they are labeled “organic,” pose a potential health threat to consumers because they are contaminated with extensively antibiotic-resistant and, presumably, virulent E. coli isolates.

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ARGONAUT II Study of the In Vitro Activity of Plazomicin against Carbapenemase-Producing Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00012-20 ◽

2020 ◽

Vol 64 (5) ◽

Author(s):

Michael R. Jacobs ◽

Caryn E. Good ◽

Andrea M. Hujer ◽

Ayman M. Abdelhamed ◽

Daniel D. Rhoads ◽

...

Keyword(s):

United States ◽

16S Rrna ◽

Klebsiella Pneumoniae ◽

Great Lakes ◽

The United States ◽

Great Lakes Region ◽

In Vitro Activity ◽

Content Type ◽

Carbapenem Resistant

ABSTRACT Plazomicin was tested against 697 recently acquired carbapenem-resistant Klebsiella pneumoniae isolates from the Great Lakes region of the United States. Plazomicin MIC50 and MIC90 values were 0.25 and 1 mg/liter, respectively; 680 isolates (97.6%) were susceptible (MICs of ≤2 mg/liter), 9 (1.3%) intermediate (MICs of 4 mg/liter), and 8 (1.1%) resistant (MICs of >32 mg/liter). Resistance was associated with rmtF-, rmtB-, or armA-encoded 16S rRNA methyltransferases in all except 1 isolate.

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Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02673-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4196-4199 ◽

Cited By ~ 22

Author(s):

Liang Chen ◽

Kalyan D. Chavda ◽

Jacqueline Findlay ◽

Gisele Peirano ◽

Katie Hopkins ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multiplex Pcr ◽

Capsular Polysaccharide ◽

Sequence Type ◽

Pcr Assay ◽

Content Type ◽

Multiplex Pcr Assay ◽

Polysaccharide Synthesis ◽

Sequence Types

ABSTRACTWe developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258]cps-1andcps-2) in epidemicKlebsiella pneumoniaeST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifyingcpstypes in 60 ST258K. pneumoniaesequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association betweencpstype andK. pneumoniaecarbapenemase (KPC) variant:cps-1is largely associated with KPC-2, whilecps-2is primarily associated with KPC-3.

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Identification of a Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae Isolate in the United States

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00519-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 11

Author(s):

Maria Karlsson ◽

Richard A. Stanton ◽

Uzma Ansari ◽

Gillian McAllister ◽

Monica Y. Chan ◽

...

Keyword(s):

United States ◽

Klebsiella Pneumoniae ◽

Outpatient Clinic ◽

The United States ◽

Sequence Type ◽

Content Type ◽

Clinical Awareness

ABSTRACT We report on a carbapenemase-producing hypervirulent Klebsiella pneumoniae (CP-hvKP) isolate collected from a U.S. patient at an outpatient clinic. The isolate was identified as K. pneumoniae serotype K1 sequence type 23 and included both a hypervirulence (with rmpA, rmpA2 iroBCDN, peg-344, and iucABCD-iutA genes) and a carbapenemase-encoding (blaKPC-2) plasmid. The emergence of CP-hvKP underscores the importance of clinical awareness of this pathotype and the need for continued monitoring of CP-hvKP in the United States.

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Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02173-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 709-711 ◽

Cited By ~ 87

Author(s):

Rong Zhang ◽

Dachuan Lin ◽

Edward Wai-chi Chan ◽

Danxia Gu ◽

Gong-Xiang Chen ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Virulence Genes ◽

Multidrug Resistant ◽

Sequence Type ◽

Zhejiang Province ◽

Content Type ◽

Carbapenem Resistant ◽

Surgical Wounds ◽

Hospital Patients

ABSTRACTWe report the emergence of five carbapenem-resistant K1 hypervirulentKlebsiella pneumoniae(hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored themagAandwcaGvirulence genes and a plasmid-borneblaKPC-2gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

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Iron Acquisition and Siderophore Release by Carbapenem-Resistant Sequence Type 258 Klebsiella pneumoniae

mSphere ◽

10.1128/msphere.00125-18 ◽

2018 ◽

Vol 3 (2) ◽

pp. e00125-18 ◽

Cited By ~ 6

Author(s):

Victoria I. Holden ◽

Meredith S. Wright ◽

Sébastien Houle ◽

Abigail Collingwood ◽

Charles M. Dozois ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Iron Acquisition ◽

Expression Patterns ◽

The United States ◽

Sequence Type ◽

Iron Limitation ◽

Transcriptional Responses ◽

Content Type ◽

Carbapenem Resistant ◽

Wide Range

ABSTRACT Klebsiella pneumoniae is rapidly acquiring resistance to all known antibiotics, including carbapenems. Multilocus sequence type ST258 (sequence type 258), carrying a gene encoding the K. pneumoniae carbapenemase (blaKPC) on a transmissible plasmid, is the most prevalent carbapenem-resistant Enterobacteriaceae (CRE) in the United States and has disseminated worldwide. Previously, whole-genome sequencing identified core genome single nucleotide variants that divide ST258 into two distinct clades, ST258a and ST258b. Furthermore, a subset of ST258b strains have a 347-base deletion within the enterobactin (Ent) exporter gene entS. Despite the predicted inability of these strains to secrete the siderophore Ent, this clade is prevalent among clinical isolates, indicating that a full-length entS gene is not necessary for infection. To compare the transcriptional responses of ST258 subtypes to iron limitation, we performed transcriptome sequencing (RNA-Seq) in minimal medium alone or supplemented with iron or human serum and measured gene expression patterns. Iron limitation induced differential expression of distinct iron acquisition pathways when comparing ST258a and ST258b strains, including the upregulation of the hemin transport operon in entS partial deletion isolates. To measure how K. pneumoniae strains vary in iron chelation and siderophore production, we performed in vitro chrome azurol S (CAS) and Arnow assays as well as mass spectrometry. We determined that both ST258a and ST258b strains grow under iron-depleted conditions, can utilize hemin for growth, and secrete Ent, despite the partial entS deletion in a subset of ST258b strains. All carbapenem-resistant (CR) K. pneumoniae strains tested were susceptible to growth inhibition by the Ent-sequestering innate immune protein lipocalin 2. IMPORTANCE Carbapenem-resistant Enterobacteriaceae, including K. pneumoniae, are a major health care concern worldwide because they cause a wide range of infection and are resistant to all or nearly all antibiotics. To cause infection, these bacteria must acquire iron, and a major mechanism of acquiring iron is by secreting a molecule called enterobactin that strips iron from host proteins. However, a subset of carbapenem-resistant K. pneumoniae strains that lack a portion of the entS gene that is required for enterobactin secretion was recently discovered. To understand how these mutant strains obtain iron, we studied their transcriptional responses, bacterial growth, and enterobactin secretion under iron-limited conditions. We found that strains both with mutated and intact entS genes grow under iron-limiting conditions, secrete enterobactin, and utilize an alternate iron source, hemin, for growth. Our data indicate that carbapenem-resistant K. pneumoniae can use varied methods for iron uptake during infection.

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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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