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BMC Infectious Diseases

, 19:928 | Cite as

Co-existence of virulence factors and antibiotic resistance in new Klebsiella pneumoniae clones emerging in south of Italy

  • Teresa FascianaEmail author
  • Bernardina Gentile
  • Maria Aquilina
  • Andrea Ciammaruconi
  • Chiara Mascarella
  • Anna Anselmo
  • Antonella Fortunato
  • Silvia Fillo
  • Giancarlo Petralito
  • Florigio Lista
  • Anna Giammanco
Open Access
Research article
Part of the following topical collections:
  1. Bacterial and fungal diseases

Abstract

Background

Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area.

Methods

50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes

Results

MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico β-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group.

Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis.

Conclusions

Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.

Keywords

Carbapenem-resistant Klebsiella pneumoniae Virulence factors 

Abbreviations

CR-Kp

Carbapenem-resistant K. pneumoniae

CS-Kp

Carbapenem-susceptible K. pneumoniae

NGS

Next generation sequencing

MLST

Multi-locus sequence typing

SNP

Core single nucleotide polymorphism

CC

Clonal complex

ST

Sequence type

WHO

World Health Organization

CDC

US Centers for Disease Control and Prevention

MDR

Multi drug resistant

Background

The World Health Organization (WHO), the US Centers for Disease Control and Prevention (CDC) and the UK Department of Health have indicated Klebsiella pneumoniae as one of the multi drug resistant (MDR) microorganisms constituting an immediate threat for human health [1, 2, 3]. K. pneumoniae, an opportunistic pathogen, has emerged not only thanks to its ability to accumulate multiclass antibiotic resistance determinants over time [1] but also, as widely reported, to its adeptness in causing severe community- and hospital-associated infections [4, 5, 6].

K. pneumoniae “permeability” to mobile genetic elements is a key factor for its dissemination not only with respect to the possibility of becoming resistant to antibiotics, but also of evolving towards more virulent phenotypes thanks to genes that may provide a survival benefit to microorganisms [7, 8]. However, in K. pneumoniae the relation between resistance and virulence is a complex issue since a systematic understanding of its population structure is still lacking [4, 9]. This makes it difficult to perceive the emergence of new clones, what instead could be an advantageous approach to develop epidemiological surveillance programs and avoid outbreaks, particularly of strains which have become resistant to carbapenem (carbapenem-resistant K. pneumoniae, CR-Kp) [9, 10, 11].

The existing body of research on carbapenem-resistance mechanisms suggests that the production of K. pneumoniae carbapenemase (KPC) encoded by the plasmidic gene blaKPC is the most common one and its rapid dissemination has typically been caused by the clonal expansion of clonal complex (CC) 258 strains, including ST258 and ST512 [12, 13, 14, 15, 16]. In Italy, the first KPC-positive K. pneumoniae, belonging to ST258, was isolated in Florence in 2008 [17]. Since then, the diffusion of these strains has been evident. In fact, the last European Antimicrobial Resistance Surveillance Network report has confirmed an average prevalence of CR-Kp of up to 33.9%, percentages that make Italy an endemic country for this microorganism [18, 19].

To date, although some research has been carried out on the diffusion and genetic characteristics of KPC-positive K. pneumoniae in our region (Sicily, Italy), no single study exists which has comprehensively described these strains considering all of the Hospital’s Departments for a period longer than 1 year [20, 21, 22, 23, 24].

In this study, we fully sequenced 50 K. pneumoniae strains, both carbapenem resistant and -susceptible, collected from March 2014 to March 2016 at the Laboratory of Bacteriology of the Department of Sciences for Health Promotion and Mother-Child Care “G. D’Alessandro” (Paolo Giaccone Polyclinic University Hospital, University of Palermo, Italy).

The primary aim of this study was to take a current snapshot of the distribution of K. pneumoniae in our geographic area by: 1) characterizing the virulome and resistome of CR-Kp clones; 2) assessing the extent to which virulence determinants were carried by CR-Kp and CS-Kp (carbapenem susceptible K. pneumoniae); 3) investigating the phylogenetic correlations among samples by Multilocus sequence typing (MLST) in silico and the analysis of core single nucleotide polymorphisms (SNPs).

Methods

Bacterial strains and antimicrobial susceptibility testing

Species and antimicrobial susceptibility were determined using the Becton- Dickinson Phoenix™ automated system (Becton Dickinson, Sparks, MD, USA). Resistance to carbapenem was established by interpreting the results of the antimicrobial susceptibility test on the basis of the breakpoint criteria of the European Committee on Antimicrobial Susceptibility Testing [25]. On the base of carbapenem susceptibility the 50 strains were divided in 25 isolates resistant (CR-Kp) and 25 sensitive (CS-Kp). Table 1 shows the entire clinical sample and the departments of isolation.
Table 1

K. pneumoniae CR and CS isolates, department of isolations, host disease, host age and clinical sample

CR-Kp ID

Department

host disease

host age

Sample

CS-Kp ID

Department

host disease

host age

Sample

1 R

General surgeries emergencies

sepsi

70

Blood PVC

1 S

Endocrinology and Metabolic diseases

ICU

75

Urine

2 R

Anaesthesia and resuscitation

sepsi

46

Blood CVC

2 S

Surgical Oncology

ICU

81

Urine

3 R

Anaesthesia and resuscitation

sepsi

59

Blood PVC

3 S

Nephrology and Hypertension

ICU

70

Urine

4 R

Anaesthesia and resuscitation

pneumoniae

60

Bronchoalveolar lavage

4 S

Clinical Respiratory Medicine

ICU

64

Urine

5 R

General surgeries emergencies

sepsi

36

Intra-abdominal fluid

5 S

General and Thoracic Surgery

ICU

52

Wound swab

6 R

Internal Medicine Cardioangiology

sepsi

36

Blood PVC

6 S

Geriatric medicine

infected wound

21

Urine

7 R

Cardiac surgery

sepsi

53

Blood CVC

7 S

Geriatric medicine

ICU

82

Ulcer swab

8 R

Clinical Respiratory Medicine

ICU

77

Urine

8 S

Plastic surgery

infected wound

82

Tissue

9 R

Internal Medicine

ICU

49

Urine

9 S

Clinical Respiratory Medicine

infected wound

74

Urine

10 R

Clinical Respiratory Medicine

ICU

79

Urine

10 S

Internal Medicine Cardioangiology

ICU

76

Sputum

11 R

Geriatric medicine

ICU

90

Urine

11 S

Infectious disease

pneumoniae

77

Sputum

12 R

Endocrinology and Metabolic diseases

ICU

84

Urine

12 S

Clinical Respiratory Medicine

pneumoniae

22

Bronchoalveolar lavage

13 R

Anaesthesia and resuscitation

sepsi

70

Blood PVC

13 S

NICU

pneumoniae

81

Endotracheal tube

14 R

Internal Medicine Cardioangiology

sepsi

54

CVC

14 S

Haematology and Bone Marrow Transplantation

pneumoniae

15 days

Sputum

15 R

Cardiac surgery

pneumoniae

72

Bronchoalveolar lavage

15 S

Rheumatology

pneumoniae

83

Urine

16 R

Anaesthesia and resuscitation

ICU

60

Urine

16 S

Internal Medicine

ICU

73

Sputum

17 R

Internal Medicine Cardioangiology

infected wound

38

Ulcer swab

17 S

Geriatric medicine

pneumoniae

76

Urine

18 R

Internal Medicine Cardioangiology

pneumoniae

77

Sputum

18 S

Rheumatology

ICU

36

Urine

19R

Cardiac surgery

infected wound

71

Wound swab

19 S

Clinical Respiratory Medicine

ICU

91

Sputum

20 R

Clinical Respiratory Medicine

ICU

80

Urine

20 S

General surgeries emergencies

pneumoniae

63

Liquor

21 R

General surgeries emergencies

bile infections

65

Bile

21 S

Internal Medicine Cardioangiology

Meningitidis

92

Urine

22 R

Anaesthesia and resuscitation

sepsi

70

Blood CVC

22 S

Anaesthesia and resuscitation

ICU

82

Urine

23 R

Cardiac surgery

pneumoniae

62

Sputum

23 S

Haematology and Bone Marrow Transplantation

ICU

70

Cutaneous swab

24R

General surgeries emergencies

sepsi

72

Intra-abdominal fluid

24 S

Tourism and Migration

infected wound

81

Urine

25 R

General surgeries emergencies

sepsi

54

Abscess fluid

25 S

Internal Medicine Cardioangiology

ICU

71

Urine

CVC: central venous catheter, ICU: intensive care unit, NICU: neonatal intensive care unit, PVC: Peripheral venous catheter

DNA isolation

The template DNA was prepared from bacterial colonies grown for 18 h on Blood Agar plates. Colonies were picked and suspended in 500 μl of ultra-pure DNase-free water. The suspension was harvested at 14000 rpm for 10 min. The supernatants were discarded while DNA from the pellets were extracted using the QIAmp® DNA Mini kit Qiagen (QIAGEN; Hilden, Germany), the quantity and purity of the DNA were determined using NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham).

Whole-genome sequencing

Isolate’s genomes were fully sequenced at the Scientific Department, Army Medical Center, Military Polyclinic of Rome (Italy) using the next-generation sequencing on the Illumina MiSeq platform (San Diego, CA, USA) as recommended by the manufacturer. The library sizes had peaks centered from 900 to 1000 bp.

The reads were de novo assembled into contigs using AByss, version 1.5.2 (k parameter = 63) [22]. Contigs longer than 500 bp were selected using an ad hoc script and kept for further analysis. The final assembly ranged from 44 to 414 (median: 143) contigs per sample (N50: 335,064–64,441; median: 111,289). Contigs were merged through the Minimus2 software (Sommer et al., 2007) and resulting DNA sequences were analysed for similarity using the database sequences by the Standard Nucleotide BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Illumina-generated sequence data for the whole data set of this study have been deposited at NCBI (BioProject id: PRJNA515715 and accession number SUB5047324).

Analysis of virulome and resistome

Virulome of all K. pneumoniae sample was analysed using the VirulenceFinder-1.4 tool and the Pasteur K. pneumoniae database. The resistome of carbapenem-resistant K. pneumoniae was analysed using the ResFinder-2.1 software (default identity thresholds [ID] 98%) which was provided by the Center for Genomic Epidemiology (http://www.genomicepidemiology.org) and the resources of the Pasteur MLST K. pneumoniae database.

Multilocus sequence typing (MLST) in silico

he in silico MLST analysis was made by comparing the whole-genome sequences against the K. pneumoniae alleles profiles available at http://www.pasteur.fr/mlst (Genotyping of Pathogens and Public Health, Institute Pasteur, Paris, France).

Core single-nucleotide polymorphisms (SNPs)

Phylogenetic analysis based on genome-wide single nucleotide polymorphisms (SNPs) were conducted detecting SNPs through the kSNP v2.1.2 program (k-mer = 21), which defines a SNP locus as an oligo of length k surrounding a central SNP allele [26]. Maximum likelihood tree based on the 57,766 core SNPs identified by kSNP program was visualised using the Dendroscope v3.2.10 software [27]. Strain 8S, K. pseudopneumoniae, was used as outgroup to root the tree.

Statistical analysis

Data were expressed as absolute numbers or percentages. The Chi-squared test was used to compare proportions (as appropriate). Values of p < 0.05 were considered statistically significant. The statistical analysis were performed with MedCalc Statistical software version 16.8 (MedCalc Software bvba, Ostend, Belgium; https://www.medcalc.org; 2016).

Results

CR-Kp were mainly isolated from blood and urinary samples (28% for both) (Fig. 1), while the ward from which CR-Kp was primarily isolated was the Anaesthesia and Resuscitation Department (24%) (Table 1). Regarding CS-Kp, urines were the major isolation sample (52%) (Fig. 1), while the ward from which CS-Kp was primarily isolated was the Respiratory Department (52%) (Table 1).
Fig. 1

Samples from where isolated the strains

Antibiotic resistance

The percentages of antibiotic resistance among K. pneumoniae carbapenem-resistant and carbapenem-susceptible strains are reported in Table 2. As shown, the CR-Kp group displayed a higher percentage of resistance for all tested antibiotics compared to the susceptible group. Statistical significance was calculated where applicable. In particular, 20% of CR-Kp and 4% of CS-Kp were colistin-resistant. Complete antibiotic resistance profile of CR-Kp are shown in Additional file 1.
Table 2

Percentage of antibiotic resistance in carbapenem resistant and susceptible K. pneumoniae

Class

Antibiotics

K. pneumoniae CR %

K. pneumoniae CS %

P value

Aminoglicosydes

Gentamycin

64

48

0.022

Carbapenems

Imipenem

100

0

NA

Meropenem

100

0

NA

Ertapenem

100

0

NA

Monobactams

Aztreonam

100

60

NA

Fluoroquinolones

Ciprofloxacin

100

64

NA

Sulfonamides-

Trimethoprim

Trimethoprim-sulfamethoxazole

76

60

0.015

Penicillin

Amoxicillin/ clavulanic acid

100

56

NA

Piperacillin/tazobactam

100

44

NA

Cephalosporin

Cefotaxime

100

60

NA

Cefuroxime

100

60

NA

Cefepime

88

56

0

Ceftazidime

100

60

NA

 

Fosfomycin c/G6P

36

16

0.001

Tetracyclin

Tigecyclin

8

4

0.233

Colistin

 

20

4

0

NA: chi-squared test not applicable

MLST analysis and Core single-nucleotide polymorphisms (SNPs) phylogenetic analysis

MLST in silico analysis of CR-Kp revealed that 52% belonged to CC258. In particular, 5 strains were ST258 (20%) and 8 were ST512 (32%). The remaining 12 strains were distributed as follows: 3 were ST395 (12%), 3 were ST307 (12%), 2 were ST392 (8%), 2 were ST348 (8%), 1 was ST405 (4%) and 1 was ST101 (4%). Among CS-Kp’s we detected 14 different STs. ST405 was the most represented (5 strains, 20%), followed by ST392 and ST15 (3 strains, 12%), ST395, ST307 and ST1727 (2 strains, 8%), and one strain each for all other STs. Figure 2 shows ST distribution across the entire sample.
Fig. 2

Percentage of ST found

The core-SNP phylogenetic analysis showed that strains belonging to the same ST clustered in the same groups, without regard to CR susceptibility or resistance (see Additional file 3). Moreover, the SNP analysis allows distinguishing each different strain.

Virulome: CR-Kp group versus CS-Kp group

Virulence factors distribution in CR-Kp and CS-Kp is detailed in Table 3. The mrk operon, which encodes type 3 fimbriae, was detected in all isolates. The wzi gene, involved in the capsule attachment to the host cell surface and used for the prediction of capsular (K) antigen type, was found in all CR-Kp isolates (100%), in particular, 3 strains carried the wzi-2 allele (see Additional file 4). Regarding the iron acquisition systems, the distribution of genes encoding for the yersiniabactin system (ybt), its receptor fyuA and the aerobactin system did not show significant differences between CR-Kp and CS-Kp (Table 3). Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group (28%) compared to the CR-Kp one (4%) (Table 3).
Table 3

Distribution of virulence factors in carbapenem resistant and susceptible K. pneumoniae strains

Virulence factors

Locus-Genes

K. pneumoniae CR

n (%)

K. pneumoniae CS

n (%)

P value

Type 3 fimbries

mrk operon

25 (100%)

25 (100%)

1.000

Capsule

wzi

25 (100%)

23 (92%)

0.552

Iron acquisition systems

ybt operon

10 (40%)

15 (60%)

0.089

Aerobactin iron acquisition siderophore

(iucABCD)

3 (12%)

3 (12%)

1.000

Klebsiella

Ferric ionic-uptake system

(kfuABC)

1 (4%)

7 (28%)

0.020

Two-component system

kvgAS

1 (4%)

7 (28%)

0.020

Bacteriocin

Microcin E492

1 (4%)

7 (28%)

0.020

Table 4 displays the comparative analysis of virulence determinants between CR-Kp and CS-Kp belonging to the same ST. All strains belonging to ST405, regardless of their resistance profile, had the same determinants (mrk, wzi-143, ybt, microcin E495 and kvgAS. Regarding the strains belonging to ST307, both CR-Kp and CS-Kp strains had the mrk operon, wzi-173 and ybt operon, with the exception of the 5R isolate, lacking ybt genes (Table 4).
Table 4

Distribution of virulence determinants in STs clone of K. pneumoniae CR and CS

STs

CR-Kp

CS-Kp

ST395

mrk, wzi-2, ybt, yuc,

mrk, wzi-2, ybt, yuc

ST307

mrk, wzi-173 ybt 1

mrk, wzi-173, ybt

ST392

mrk, wzi-187

mrk, wzi-187

ST405

mrk, wzi-143, ybt, kvgAS, E495

mrk, wzi-143, ybt, kvgAS, E495

1missing in one of the three CR isolates

Virulome subanalysis by STs across the CR-Kp group

CR-Kp isolates belonging to ST512, ST258 did not carry virulence determinants other than mrk and wzi (Table 4). The three strains belonging to ST395 carried four virulence determinants: mrk, fyuA/irp2, iucABCD and wzi type 2. wzi gene type 27 was found in the two ST392 isolates together with the mrk operon. Strains belonging to ST348 carried the mrk operon, ybt operon and wzi-94 gene, whereas the ST101 strain exhibited mrk, ybt operon, wzi type 17 and the kfuABC system. The ST405 isolate was the only one that carried the kvgAS operon and the microcin E495, together with the mrk operon, ybt operon and the wzi-143.

Resistome analysis across the CR-Kp group

The in silico β-lactamase characterisation of CR-Kp isolates showed that the most frequent carbapenemase-producing gene was blaSHV (100%). In particular, SHV variant 182 was detected in 16 out of 25 isolates (64%), while variant 28 in four strains (16%), three ST307 and the only ST101 isolate. blaKPC was identified in 88% of isolates and the most common variant was blaKPC-3 (90.9%). blaTEM was found in 23 isolates (92%), all of which were variant 1 and blaCTX-M (variant 15) was found in 8 isolates (32%). blaOXA was found in 22 isolates (88%), 14 of these were variant 9 (63.6%), 5 were variant 1 (22.72%) and 3 isolates (13.63%) presented both variants, blaOXA-1 and blaOXA-9. Five isolates, one belonging to ST395, two to ST307, one to ST101 and one to ST405 owned all the five carbapenemase-producing genes investigated (Table 5). Complete data from the in silico analysis (e.g. encoding efflux pumps, heavy metal resistance system, genes involved in aminoglycoside and fluoroquinolone resistance) are shown in Additional file 2.
Table 5

K. pneumoniae CR profile: colistin susceptibility, carbapenemase, ESBL and beta-lactamases genes

STs

ID

CS (mg/L)

bla KPC

bla SHV

bla CTX-M

bla TEM

bla OXA

ST512

6 R

<=1

3

182

1

9

7 R

<=1

3

182

1

9

8 R

<=1

3

182

11 R

<=1

3

182

1

9

16 R

<=1

3

182

1

9

19 R

> 4

3

182

1

22 R

<=1

3

182

1

9

25 R

<=1

3

182

1

9

ST258

12 R

<=1

3

182

1

9

18 R

<=1

3

182

1

9

21 R

<=1

3

182

1

9

23 R

> 4

3

182

1

9

24 R

<=1

3

182

1

9

ST395

2 R

<=1

3

182

1

4 R

<=1

3

182

15

1

1

10 R

<=1

3

182

1

ST307

5 R

<=1

9

28

15

1

9

13 R

<=1

3

28

15

1

1/9

20 R

> 4 R

2

28

1

1/9

ST392

1 R

<=1

3

67

15

1

9

3 R

<=1

67

15

1

1

ST 348

15 R

> 4 R

81

15

1

1

17 R

<=1

81

15

1

1

ST 101

9 R

<=1

3

28

1

9

ST 405

14 R

> 4 R

3

76

15

1

1/9

Discussion

The epidemiology of CR-Kp in our geographic area (Palermo, Italy) has already been characterized in the late 2008 at the emergence of CR-Kp ST258 clones [20]. However, several reports have suggested an ongoing epidemiological change in the last years. In fact, whereas CC258 (ST258 and ST512) is still prevalent, several other STs are emerging and circulating [23, 24, 28].

This study set out with the aim of assessing the current dissemination and genetic characteristics of K. pneumoniae in Palermo. Even though a larger sample may have allowed to gain more representative data, our preliminary data reveal a complex situation characterized by: 1) high genome “plasticity” of both CR-Kp and CS-Kp, due to the presence of several virulence and resistance determinants carried by mobile genetic elements; 2) a CR-Kp group showing an important genetic diversity of lineages, with 8 different STs identified; 3) an overlapping of multi-drug resistance and hyper virulence traits in the CR-Kp group.

Regarding STs, our comprehensive analysis indicates that, although ST258 and ST512 remain the most representative ones, other STs (e.g. ST307, ST395, ST392, ST348, ST405 and ST101) have been detected in our area. These results are consistent with the surveillance data from other authors [20, 21, 22, 23, 24, 28]. Moreover, our findings on β-lactamase characterisation showed that the blaSHV gene was the most commonly found in our sample, followed by blaTEM and blaKPC, deviating from other studies [11, 29, 30], while respect to the KPC-type enzyme our results are in line with those of other studies - as the most commonly encountered is blaKPC-3 [21, 23, 31, 32, 33]. Regarding STs, the most represented across the CR-Kp group were ST512 and ST258. These isolates did not carry any distinguishing virulence determinant (except for the mrk operon and wzi gene, which were present in all the samples), suggesting that the success of these clones may only depend on the acquisition of the blaKPC gene [9, 15, 34].

Among the ST512 isolates, we found the 19R strain profile to be particularly interesting. This colistin-resistant strain was the only one in its ST group that showed both the yersiniabactin system and its receptor, which has been detected in several K. pneumoniae MDR clones; despite of this, the clinical effect of the yersiniabactin system on CR-Kp infections has not been clearly determined [4, 35, 36].

Our data also match those of reports suggesting the recent spread of the well-known clone ST307. In fact, in 2014, a CR-Kp ST307 clone carrying the blaKPC-3, coproducing the blaCTX-M-15, has been isolated in three Palermo’s Hospitals [23]. The virulome analysis of our ST307 strains has revealed, that 5R isolate was the only one missing the yersiniabactin locus in its ST group. Furthermore, 13R and 5R isolates, two of the three strains belonging to ST307, showed the co-presence of all five carbapenem resistance genes analysed. The co-presence of the detected virulence factors together with the MDR phenotype may explain the diffusion of this clone and the severity of its infections, which have been reported as characterised by higher mortality rates (over 50%) compared to other clones [37, 38]. Moreover, leaving aside specific considerations about the clone ST307, the co-presence of five carbapenem resistance genes that we detected in five strains of our sample (20%) is in line with that of a study by Ferreira et al., which has recently reported that 72% of the K. pneumoniae isolated from a Brazilian Intensive care Unite co-produced blaKPC, blaOXA, blaTEM, blaSHV, and blaCTX-M [39].

Another emerging clone, already isolated by other authors in Palermo and also detected in our CR-Kp sample, was the ST395 [40]. Strains belonging to this clone presented the wzi2 allele, which encodes the type K2 capsular antigen that represents one of the most virulent serotypes, thus defined “more virulent” [41, 42]. CR-Kp ST395 strains also carried the yersiniabactin system and its receptor and were the only resistant isolates to possess the aerobactin system (iucABCD). Two aspects of these strains should be addressed here: i) 4R isolate showed the co-presence of five carbapenem resistance genes; ii) to the best of our knowledge, this is the first time that a CR-Kp ST395 clone is reported as carrying a type 2 capsule. This finding was unexpected and seems to be in contrast with the concept that MDR and hyper virulent clonal complexes do not normally overlap [42].

Moreover, our data confirm the spreading of the ST392 clone. A recently published study by Di Mento and colleagues has reported, for the first time, the isolation of a K. pneumoniae strain ST392 blaKPC-3 carrying the blaCTX-M-15, blaSHV-11 and blaTEM-1 genes from a patient in Palermo who had undergone kidney-pancreas transplantation [43]. In our CR-Kp ST392 sample, the 1R strain which was isolated from blood at the General and Emergency Surgery Department in 2015, showed the co-existence of blaKPC-3, blaSHV-67, blaCTX-M-15, blaTEM-1 and blaOXA-9. This result is interesting as the only other KPC-producing K. pneumoniae ST392 ever reported was isolated in China but with a different isoform, KPC-2 [44]. However, it is important to underline that the other ST392 strain (3R) in our CR-Kp sample did not carry the blaKPC gene, suggesting that the ST392 KPC-3 clone may have acquired the resistance gene through horizontal transmission, as described by other authors [43].

This study also revealed two MDR CR-Kps belonging to ST348 and carrying the blaCTX-M-15, blaSHV-81, blaTEM-1 and blaOXA-1 genes. Strains belonged to ST348, but harboured the blaKPC-3, which had been previously reported as responsible of several epidemic events in Portugal [45]. Considering that one of our isolates was colistin-resistant and the ease with which K. pneumoniae acquires the blaKPC gene, we can consider the MDR CR-Kp ST348 strains as possibly emerging high-risk clones.

Three other important data that emerge from our results and complete the description of the CR-Kp epidemiological scene in Palermo are: firstly, the circulation of strains belonging to ST101. This clone was previously recognized worldwide as a high risk carbapenem-producing clone [30] and has already been identified in Palermo and in the North of Italy [46, 47, 48]. Our ST101 strain (9R) carried the blaKPC-3, blaSHV-28, blaTEM-1 and blaOXA-9 genes and the Klebsiella ferrous uptake system, which is typically found in K. pneumoniae hypervirulent strains [49, 50]; secondly, the characteristic of the strain belonging to ST405, which carried the blaKPC-3, blaCTX-15, blaSHV-76, blaTEM-1 and blaOXA-1/9 genes, the aac6-Ib-cr and qnrB and was resistant to colistin. Strains from ST405 have similarly caused an outbreak in a Spanish Hospital neonatal unit [50] and have already been isolated in Palermo [24]. It is important to underline that the genes involved in microcin production and kvgA/S system were detected in all isolates belonging to ST405, both resistant and susceptible to carbapenem, possibly indicating a stable and characteristic genetic pattern for these clones; lastly to the best of our knowledge this is the first study to report in Italy the isolation of five CR-Kp isolates belonging to different STs showing the co-presence of five carbapenem resistance genes.

Conclusions

These results are significant in at least two major respects. Overall, this study strengthens the idea that the epidemiological frame in the Palermo area (Sicily, Italy) is shifting and new MDR clones are emerging. However, our analysis, which included the comparison of the virulence degree of CS-Kp and CR-Kp isolates, has unexpectedly revealed that the latter are acquiring highly-virulent determinants and the co-presence of more resistance genes. Undoubtedly, since co-existence of antibiotic resistance and virulence factors may lead to life-threatening untreatable and invasive K. pneumoniae infections, this is an important issue to take into consideration for future genomic surveillance studies.

Notes

Acknowledgements

We should like to express our gratitude to SciencED medical communication for its technical support.

Consent to publish

Not applicable.

Authors’ contributions

Conceived and designed the study: TF, AG, FL. Managed the conduction of the study: TF, AG, FL, MA. Collected samples: CM, MA, TF. Microbiological analysis and molecular typing: BG, AA, AF, SF, GP . Analysed the data: BG, AA, AF, SF, GP, AC. Drafted the manuscript: AG, TF, MA, FL. Drafted the revisions: AC, TF. All authors read and approved the final manuscript.

Funding

The authors received no specific funding for this work.

Ethics approval and consent to participate

All examined carbapenem-resistant Klebsiella pneumoniae and carbapenem-sensible Klebsiella pneumoniae isolates were isolated during the routine diagnostics and preserved according to local epidemiological surveillance regulations. Written informed consent for routine diagnostic and medical procedures was collected for each patient.

All data used in the study were anonymized, according to the requirements set by Italian Data Protection Code (leg. Decree 196/2003) and by the general authorizations issued by the Data Protection Authority. Approval by the Ethics Committee was obtained by Azienda Ospedaliera Universitaria Policlinico “P. Giaccone” of Palermo (protocols n°07/2019).

Competing interests

The author declare that they have no competing interests.

Supplementary material

12879_2019_4565_MOESM1_ESM.docx (31 kb)
Additional file 1. K. pneumoniae CR antibiotic resistance profile. Results of antibiotic resistance assay of K. pneumoniae CR.
12879_2019_4565_MOESM2_ESM.docx (35 kb)
Additional file 2. K. pneumoniae CR in silico analysis of resistome and virulome. Results of in silico analysis of sequences encoding for efflux pumps, heavy metal resistance system, and genes involved to aminoglycoside and fluoroquinolone resistance.
12879_2019_4565_MOESM3_ESM.docx (105 kb)
Additional file 3. wzi analysis of CR-K and CS-K. Table of contig and allele of wzi gene in K. pneumoniae CR and CS.
12879_2019_4565_MOESM4_ESM.docx (32 kb)
Additional file 4. SNP phylogenetic tree. Core Single-Nucleotide Polymorphisms dendrogram.

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Teresa Fasciana
    • 1
    Email author
  • Bernardina Gentile
    • 2
  • Maria Aquilina
    • 1
  • Andrea Ciammaruconi
    • 2
  • Chiara Mascarella
    • 1
  • Anna Anselmo
    • 2
  • Antonella Fortunato
    • 2
  • Silvia Fillo
    • 2
  • Giancarlo Petralito
    • 2
  • Florigio Lista
    • 2
  • Anna Giammanco
    • 1
  1. 1.Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical SpecialtiesUniversity of PalermoPalermoItaly
  2. 2.Scientific Department, Army Medical CenterRomeItaly

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