- Email: [email protected]
Klebsiella pneumoniae KPC producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene
Journal Pre-proof Klebsiella pneumoniae KPC producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene Elisa Antinori, Ilaria Unali, Anna Bertoncelli, Annarita Mazzariol PII:
S1198-743X(20)30086-0
DOI:
https://doi.org/10.1016/j.cmi.2020.02.007
Reference:
CMI 1930
To appear in:
Clinical Microbiology and Infection
Received Date: 3 November 2019 Revised Date:
2 February 2020
Accepted Date: 6 February 2020
Please cite this article as: Antinori E, Unali I, Bertoncelli A, Mazzariol A, Klebsiella pneumoniae KPC producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2020.02.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
Klebsiella pneumoniae KPC producer resistant to ceftazidimeavibactam due to a deletion in the blaKPC3 gene Elisa Antinori, Ilaria Unali, Anna Bertoncelli, Annarita Mazzariol Department of Diagnostics and Public Health, University of Verona, Verona, Italy
Corresponding author: Annarita Mazzariol Department of Diagnostics and Public Health University of Verona Strada Le Grazie, 8 37134 Verona, Italy Phone: +39 045 8027690 Fax: +39 045 8027101 Email: [email protected]
1
2
Keywords:
ceftazidime-avibactam
resistance;
3
carbapenemase, KPC deletion, KPC mutations
KPC
producing
K.
pneumoniae,
KPC
4 5
Abstract
6
Objectives: Klebsiella pneumoniae producing KPC is a great health concern and therapy with
7
ceftazidime-avibactam represent a choice for the treatment of infections supported by these strains.
8
We report a ceftazidime-avibactam resistant strain by a deletion of 6 nucleotides in the blaKPC gene
9
sequence.
10
Materials/methods: two strains, namely AMP920 and AMP2009 were isolated from same patient a
11
month away. Antimicrobial susceptibility was performed both by broth microdiluition and Etest.
12
Immune-enzymatic assay to detect carbapenemase was performed for both strains. blaKPC gene of
13
both strains were amplified by PCR and sequenced. Enzyme activity towards carbapenems was
14
tested by CarbaNP test and hydrolysis spectrophotometer assay.
15
Results: The two isolates differ in antimicrobial susceptibility testing. AMP920 show meropenem
16
and imipenem resistance (MIC 32 and 32 mg/ml, namely). A month later carbapenems MIC
17
decreased to 8 and 1 mg/ml respectively, while ceftazidime-avibactam MIC increase from 1 to 16
18
mg/ml. Both isolates showed a positive immune-enzymatic test for KPC enzyme, but only AMP920
19
showed positive CarbaNP test hydrolyzing imipenem. BlaKPC gene was amplified in both strains.
20
After sequencing the two amplicons showed a KPC3 variant. The gene of second isolate showed a
21
deletion of 6 nucleotides in position 498-503, bringing a mutate variant with the following deletion
22
of a glutamic acid and leucine in position 167 and 168.
23
Conclusions: we detect a new deletion in the blaKPC gene of a clinical strain bringing to
24
ceftazidime-avibactam resistance. The amino acids deleted are in in the omega-loop (amino acids
25
165 through 179) of KPC enzyme, enhancing ceftazidime affinity preventing avibactam binding.
26 27
Introduction
28
Along with carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas
29
aeruginosa, carbapenem-resistant Enterobacteriaceae (CRE) are considered antibiotic-resistant
30
“priority pathogens” by WHO. These bacteria represent pivotal threats to human health [1].
31
Enterobacteriaceae are common pathogens causing a variety of severe infections, including
32
bloodstream infections, community-acquired pneumonia, hospital acquired pneumonia, ventilator-
33
associated pneumonia, complicated urinary tract infections, and complicated intra-abdominal
34
infections. Hence, the onset of antibiotic resistance in these bacteria determines significant clinical
35
and socioeconomic impacts [2].
36
Klebsiella pneumoniae producing KPC carbapenemase is an important health concern. Majority of
37
the strains isolated in Italy belong to the clonal complex ST258 and our country is considered
38
endemic for KPC-producing K. pneumoniae [3].
39
To date, the treatment options for CRE infections are very limited. Polymyxins (colistin or
40
polymyxin B) and tigecycline have been historically considered as drugs of choice for infections
41
caused by CRE. However, resistance to these antibiotics is increasing [4].
42
Avibactam is a non-beta-lactam beta-lactamase inhibitor directed to class A, class C and some class
43
D beta-lactamases. FDA has approved, in 2015, the application of this compound in combination
44
with ceftazidime for treating infections caused by antibiotic resistant pathogen. Ceftazidime-
45
avibactam therapy represents a precious approach for the treatment of infections supported by these
46
strains [5].
47
Several observations have reported that the mechanisms of resistance or low susceptibility to
48
ceftazidime-avibactam are related to evolution of isoform of blaCTX-M-14 or even CTX-M1 family
49
with a higher ceftazidime hydrolysing capacity [6] or mutations in the Ω-loop of KPC β-lactamases,
50
for example D179Y in KPC-3 [7, 8].
51
The aim of present study is to investigate the resistance mechanism to ceftazidime-avibactam
52
observed in an infected patient with a K. pneumoniae blaKPC producing strain.
53
Materials and methods
54
Two K. pneumoniae strains, namely AMP920 and AMP1009 were isolated from same patient on
55
half September and late October 2018 respectively, from surgical wound swabs during routine
56
clinical examinations and no human subjects were involved in the study. Antimicrobial
57
susceptibilities were performed with Vitek2 (Biomèrieux, France) and subsequently confirmed both
58
by broth micro-dilution and Etest. Antibiotics tested were carbapenems (Ertapenem, Meropenem
59
and Imipenem), cephalosporin (Ceftazidime and Cefotaxime), Colistin and Ceftazidime-Avibactam.
60
Carbapenemase activity was first checked by CarbaNP test [9] and after was confirmed by
61
hydrolysis test of raw protein extract. Carbapenemase enzyme type was investigated by the NG-
62
Test Carba 5 (Biotech), an immune-chromatographic assay which use a multiplex Lateral Flow
63
Immune Assay (LFIA) to detect NDM, OXA-48, KPC, VIM and IMP-like and blaKPC gene was
64
amplified by PCR [10] and sequenced (MWG Operons, Germany). ESBL genes (blaCTX-M, blaTEM
65
and blaSHV) were investigated too by PCR [11].
66
Genetic relatedness of the two strains was determined by MLST (Pasteur protocol,
67
https://pubmlst.org/kpneumoniae/) and PFGE.
68
KPC detection by 11,109 Da peak analysis of Maldi-tof spectra was also performed [12-13].
69
Presence of Tn4401 and pO19 elements associated with blaKPC resistance gene have been detected
70
with a PCR reaction [13].
71
PCR Based Replicon Typing (PBRT) protocol [14] was performed to check the plasmid profile of
72
the strains.
73 74
Results
75
Antimicrobial susceptibilities results for both strains are reported in Table 1. The first isolate
76
AMP920, showed meropenem and imipenem resistance, whit MIC of 8 mg/L. The second isolate
77
AMP1009, an isolate later from same patient showed a meropenem MIC of 1 mg/L, while
78
ceftazidime-avibactam MIC between the two isolates change from 1 to 16 mg/L. When AMP920
79
was isolated, since it showed a carbapenem resistant phenotype the strain was checked for
80
carbapenemase activity by CarbaNP test [9] that resulted positive. Carbapenemase enzyme type was
81
investigated by immuno-chromatographic assay (NG-Test CARBA5, NG Biotech) showing the
82
presence of KPC. The second isolate showed an ESBL producer phenotype by Vitek2 system. Since
83
the patient previously presented a K. pneumoniae KPC producer, the second isolate AMP1009 was
84
further investigated. Carbapenem and Ceftazidime-avibactam MICs were checked by Etest first and
85
then confirmed with broth micro-dilution. Also for the AMP1009 isolate CarbaNP and immune-
86
chromatographic test were performed. CarbaNP test for this second isolate resulted negative while
87
the immune-chromatographic test showed the production of KPC enzyme (results summarized in
88
Table 1). Absence or presence of carbapenemase activity was confirmed also by hydrolysis test of
89
raw protein extract and no enzyme able to hydrolyse carbapenems was reported in the AMP1009.
90
To solve this discrepancy, blaKPC gene of both strains were amplified by PCR [10] and sequenced.
91
The sequencing was able to identify a KPC3 variant in the two amplicons. As reported in Figure 1,
92
the blaKPC gene of AMP1009 isolate showed a deletion of 6 nucleotides in position 498-503,
93
bringing a mutate variant with the following deletion of a glutamic acid and leucine in position 167
94
and 168. The complete blaKPC gene sequencing was performed, to investigate genetic differences.
95
The BLAST alignment revealed that blaKPC genes of two isolates matched with the well-known
96
carbapenem-hydrolyzing class A beta-lactamase KPC-3 gene.
97
This mutation affects the proton acceptor active site located in position 167, corresponding to one of
98
the two amino acids deleted. The UniProt analysis of the carbapenem-hydrolyzing β-lactamase KPC
99
(UniProtKB - Q9F663 (BLKPC_KLEPN)), gave us some important informations about where the
100
active sites were located into protein structure and, in detail, about the proton acceptor one. The
101
latter, composed by a glutamic acid (LDRWELELNS), is located at position 167, exactly the site of
102
deletion.
103
BlaSHV, blaTEM were also detected by PCR in both strains.
104
Genetic relatedness of the two strains was determined by MLST (Pasteur protocol) and PFGE. Both
105
strains showed the same MLST profile, corresponding to ST512, belonging to CC256. The clonally
106
of the two isolates was also confirmed by PFGE. The two isolates show a correlation amid 95% and
107
100%, suggesting that those two isolates belong to the same clonal lineage.
108
The peptide mass fingerprint (PMF) generated by MALDI-TOF MS analysis revealed the presence
109
of a 11 109Da peak in both the isolates [10, 11]. This peak is associated to the protein pO19
110
inserted into the same region of the transposon tn4401, often harbouring the blaKPC gene in K.
111
pneumoniae carbapenemase producers' strains. The Tn4401a isoform and pO19 elements associated
112
with blaKPC resistance gene have been detected with a PCR reaction [13].
113
Following the PBRT protocol [14] both the isolates harbour the FIIs plasmid, a pKpQIL-like
114
plasmid often associated with the blaKPC gene.
115
Discussion
116
In our report, we have described a new mechanism of ceftazidime-avibactam resistance with
117
decreasing activity against carbapenems in a K. pneumoniae strain KPC producer in an infected
118
patient. The mechanism of resistance to ceftazidime-avibactam is due to a deletion of 6 nucleotides
119
in position 498-503, that brings to a new variant with a deletion of two amino-acids in position 167-
120
168 The amino-acid in position 167 is involved in the proton acceptor active site.
121
Livermore et al [8] reported the vulnerabilities of ceftazidime-avibactam to select spontaneous
122
mutations bringing to resistance. Winkler et al. [15] reported the activity of ceftazidime-avibactam
123
against a set of isogenic E. coli strains with mutation in the omega-loop of KPC enzyme,
124
demonstrating that substitution of the amino acid positions 165 through 179, enhanced ceftazidime
125
affinity preventing avibactam binding. Our clinical isolate with a deletion of amino acid 167-168 of
126
KPC-3 variant in K. pneumoniae seems to confirm in vivo the importance of mutations in this site to
127
confer ceftazidime-avibactam resistance.
128
An important limitation of this study is the lack of information about patient treatment. Our data
129
support the hypothesis that this type of mutation could be selected by therapy. Now we are planning
130
to investigate on our ceftazidime-avibactam resistant strains and the ceftazidime-avibactam therapy.
131
Another limitation is the lack of characterization of biochemical property such the hydrolytic
132
activity of this variant compare with the KPC-3, even if we know that KPC-3 variant harbored in
133
FII-like plasmid in strains isolated in our hospital setting [ 16] has a size of 97 Kb determined by
134
S1-PFGE and trans conjugant E. coli JC53 showed a meropenem MIC of 1 mg/L.
135
Our report underline also the difficulties of clinical laboratories to reveal these ceftazidime-
136
avibactam resistant isolates since most of them, like in this case were reported as meropenem
137
susceptible, ESBL producing and not screened and identified as KPC producing strain. Also
138
molecular blaKPC detection or immune-chromatographic KPC screening are not able to reveal KPC
139
variants with different affinities and hydrolytic properties.
140
In order to preserve ceftazidime-avibactam activity and monitor its resistance we think that is
141
necessary to test this antibiotic combination not only in presence of a class A carbapenemase but for
142
all strains that present an ESBL phenotype. This assume more importance when patients are known
143
previously colonized or infected with carbapenemase producing Enterobacteria and the new isolates
144
show a carbapenems susceptibility since this may hide a ceftazidime-avibactam resistance.
145 146
Authors’ contribution
147
Elisa Antinori: Investigation, Formal analysis, writing original draft
148
Ilaria Unali; Investigation, validation
149
Anna Bertoncelli: Resources, validation
150
Annarita Mazzariol: Conceptualization, Data curation, writing review-editing, supervision
151
Funding: No external funding was received
152
Conflict of interests: None
153
Acknowledgement: Results were partially presented at the 29th ECCMID, took place in
154
Amsterdam Netherlands, 13-16 April 2019
155 156
References
157 158 159
1. Willyard C. The drug-resistant bacteria that pose the greatest health threats. Nature 2017;543(7643):15
160
2. Rodriguez-Bano J., Gutierrez-Gutierrez B., Machuca I., and Pascual A. Treatment of
161
infections caused by extended-spectrum-beta-lactamase-, AmpC, and carbapenemase-
162
producing Enterobacteriaceae. Clin. Microbiol. Rev. 2018: 31: e00079-17.
163
3. Giani T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R et al. Epidemic diffusion of
164
KPC carbapenemase producing Klebsiella pneumoniae in Italy: results of the first
165
countrywide survey. 15 May to 30 June 2011. Eurosurveillance 2013: May 30;18 (22) pii:
166
20489.
167
4. Wang Q., Zhang P., Zhao D., Jiang Y., Zhao F., Wang Y., et al. Emergence of tigecycline
168
resistance in Escherichia coli co-producing MCR-1 and NDM-5 during tigecycline
169
salvage treatment. Infect. Drug Resist. 2018: 11 2241–2248.
170
5. Sheu CC, Chang YT, Lin SY, Chen YH, Hsueh PR. Infections Caused by Carbapenem-
171
Resistant Enterobacteriaceae: An Update on Therapeutic Options. Front Microbiol. 2019
172
Jan 30;10:80.
173
6. Both A, Butter H, Huang J, Perbandt M, Belmar Campos C et al. Emergence of
174
ceftazidime/avibactam non-susceptibility in an MDR Klebsiella pneumoniae isolate. J.
175
Antimicrob Chemother 2017:72:2483-88.
176
7. Haidar G, Clancy CJ, Shields RK, Hao B, Cheng S, Nguyen MH. Mutations in blaKPC-3
177
That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That
178
Function as Extended-Spectrum beta-lactamases. Antimicrob Agents Chemother. 2017:6:
179
e02534-16.
180
8. Livermore DM, Warner M, Jamrozy D, Mushtaq S, Nichols WW, Mustafa N, Woodford
181
N. In vitro selection of ceftazidime-avibactam resistance in Enterobacteriaceae with
182
KPC-3 carbapenemase. Antimicrob. Agents Chemother. 2015.59:5324 –5330.
183
9. Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase producing
184 185
Enterobcateriaceae. Emerg Inf. Dis 2012:18:150-7. 10. Mazzariol A, Lo Cascio G, Ballarini P, Ligozzi M, Soldani F, Fontana R et al. Rapid
186
molecular technique analysis of a KPC-3-producing Klebsiella pneumoniae outbreak in an
187
Italian surgery unit. J.Chemother». 2012:24:93-6.
188
11. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex
189
PCR assays for the detection of genes encoding important β-lactamases in
190
Enterobacteriaceae. J Antimicrob Chemother 2010: 65: 490–495.
191 192
12. Lau AF, Wang H, Weingarten RA, Drake SK, Suffredini AF et al. A Rapid MatrixAssisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method
193
for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant
194
Enterobacteriaceae. J. Clin. Microb. 2014; 52 (8): 2804–2812.
195
13. Centonze AR, Bertoncelli A, Savio C, Orza P, Bedenic B, Mazzariol A. Evaluation of a
196
rapid KPC carbapenemase detection method based on MALDI-TOF spectra analysis. J.
197
Med. Microbiol. 2018: 67: 1474-1479. Epub 2018 Jan 12.
198 199
14. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–228.
200
15. Winkler ML, Papp-Wallace KM, Bonomo RA. Activity of ceftazidime/avibactam against
201
isogenic strains of Escherichia coli containing KPC and SHV beta-lactamases with single
202
amino acid substitutions in the Omega-loop. J Antimicrob Chemother 2015. 70:2279
203
16. Kocsis E., Lo Cascio G, Piccoli M, Cornaglia G, Mazzariol A. KPC-3 Carbapenemase
204
Harbored in FIIk Plasmid from Klebsiella pneumoniae ST512 and Escherichia coli ST43
205
in the Same Patient. Microb Drug Res. 2014:20:377-82
206 207
Table 1: Antimicrobial susceptibilities and carbapenemase phenotypic tests of the two strains under study.
Strains CZA AMP920 1 AMP1009 16
MICs mg/L ERT >128
4
IMP 8 0.5
MPM 8 1
CAZ >128 >128
CTX 64 4
COL 0,5 0,5
CarbaNP
NGcarba5
pos neg
KPC KPC
CZA: ceftazidime-avibactam; ERT: Ertapenem; IMP: Imipenem; MPM: Meropenem; CAZ: ceftazidime; CTX: cefotaxime; COL: Colistin
Figure 1: Partial Nucleotide and amino acid alignments of blaKPC3 sequence and blaKPC amplicon of AMP1009 strain showing 6 nucleotides deletion. In bold and in the square are reported amino acids position involved in the KPC3 omega loop.
AMP1009 bla KPC3
…………… GTT CCG TCT GGA CCG CTG ___ ___ GGA GCT GAA CTC CGC CAT CCC AGG CGA TGC GCG CGA ………… | || | | | | || | | | | | | || | | | | | || | | | || | | | | | || | | | | | | | | | | | | | | | | | | …………… GTT CCG TCT GGA CCG CTG GGA GCT GGA GCT GAA CTC CGC CAT CCC AGG CGA TGC GCG CGA ……….
AMP1009
…………..
blaKPC3
…………..
F * F
aa position
…………..
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
R * R
L * L
D * D
R * R
W * W
_
_
E
L
E * E
L * L
N * N
S * S
A * A
I * I
P * P
G * G
D * D
A * A
R * R
D * D
…………. …………. ………….
S1198-743X(20)30086-0
DOI:
https://doi.org/10.1016/j.cmi.2020.02.007
Reference:
CMI 1930
To appear in:
Clinical Microbiology and Infection
Received Date: 3 November 2019 Revised Date:
2 February 2020
Accepted Date: 6 February 2020
Please cite this article as: Antinori E, Unali I, Bertoncelli A, Mazzariol A, Klebsiella pneumoniae KPC producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2020.02.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
Klebsiella pneumoniae KPC producer resistant to ceftazidimeavibactam due to a deletion in the blaKPC3 gene Elisa Antinori, Ilaria Unali, Anna Bertoncelli, Annarita Mazzariol Department of Diagnostics and Public Health, University of Verona, Verona, Italy
Corresponding author: Annarita Mazzariol Department of Diagnostics and Public Health University of Verona Strada Le Grazie, 8 37134 Verona, Italy Phone: +39 045 8027690 Fax: +39 045 8027101 Email: [email protected]
1
2
Keywords:
ceftazidime-avibactam
resistance;
3
carbapenemase, KPC deletion, KPC mutations
KPC
producing
K.
pneumoniae,
KPC
4 5
Abstract
6
Objectives: Klebsiella pneumoniae producing KPC is a great health concern and therapy with
7
ceftazidime-avibactam represent a choice for the treatment of infections supported by these strains.
8
We report a ceftazidime-avibactam resistant strain by a deletion of 6 nucleotides in the blaKPC gene
9
sequence.
10
Materials/methods: two strains, namely AMP920 and AMP2009 were isolated from same patient a
11
month away. Antimicrobial susceptibility was performed both by broth microdiluition and Etest.
12
Immune-enzymatic assay to detect carbapenemase was performed for both strains. blaKPC gene of
13
both strains were amplified by PCR and sequenced. Enzyme activity towards carbapenems was
14
tested by CarbaNP test and hydrolysis spectrophotometer assay.
15
Results: The two isolates differ in antimicrobial susceptibility testing. AMP920 show meropenem
16
and imipenem resistance (MIC 32 and 32 mg/ml, namely). A month later carbapenems MIC
17
decreased to 8 and 1 mg/ml respectively, while ceftazidime-avibactam MIC increase from 1 to 16
18
mg/ml. Both isolates showed a positive immune-enzymatic test for KPC enzyme, but only AMP920
19
showed positive CarbaNP test hydrolyzing imipenem. BlaKPC gene was amplified in both strains.
20
After sequencing the two amplicons showed a KPC3 variant. The gene of second isolate showed a
21
deletion of 6 nucleotides in position 498-503, bringing a mutate variant with the following deletion
22
of a glutamic acid and leucine in position 167 and 168.
23
Conclusions: we detect a new deletion in the blaKPC gene of a clinical strain bringing to
24
ceftazidime-avibactam resistance. The amino acids deleted are in in the omega-loop (amino acids
25
165 through 179) of KPC enzyme, enhancing ceftazidime affinity preventing avibactam binding.
26 27
Introduction
28
Along with carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas
29
aeruginosa, carbapenem-resistant Enterobacteriaceae (CRE) are considered antibiotic-resistant
30
“priority pathogens” by WHO. These bacteria represent pivotal threats to human health [1].
31
Enterobacteriaceae are common pathogens causing a variety of severe infections, including
32
bloodstream infections, community-acquired pneumonia, hospital acquired pneumonia, ventilator-
33
associated pneumonia, complicated urinary tract infections, and complicated intra-abdominal
34
infections. Hence, the onset of antibiotic resistance in these bacteria determines significant clinical
35
and socioeconomic impacts [2].
36
Klebsiella pneumoniae producing KPC carbapenemase is an important health concern. Majority of
37
the strains isolated in Italy belong to the clonal complex ST258 and our country is considered
38
endemic for KPC-producing K. pneumoniae [3].
39
To date, the treatment options for CRE infections are very limited. Polymyxins (colistin or
40
polymyxin B) and tigecycline have been historically considered as drugs of choice for infections
41
caused by CRE. However, resistance to these antibiotics is increasing [4].
42
Avibactam is a non-beta-lactam beta-lactamase inhibitor directed to class A, class C and some class
43
D beta-lactamases. FDA has approved, in 2015, the application of this compound in combination
44
with ceftazidime for treating infections caused by antibiotic resistant pathogen. Ceftazidime-
45
avibactam therapy represents a precious approach for the treatment of infections supported by these
46
strains [5].
47
Several observations have reported that the mechanisms of resistance or low susceptibility to
48
ceftazidime-avibactam are related to evolution of isoform of blaCTX-M-14 or even CTX-M1 family
49
with a higher ceftazidime hydrolysing capacity [6] or mutations in the Ω-loop of KPC β-lactamases,
50
for example D179Y in KPC-3 [7, 8].
51
The aim of present study is to investigate the resistance mechanism to ceftazidime-avibactam
52
observed in an infected patient with a K. pneumoniae blaKPC producing strain.
53
Materials and methods
54
Two K. pneumoniae strains, namely AMP920 and AMP1009 were isolated from same patient on
55
half September and late October 2018 respectively, from surgical wound swabs during routine
56
clinical examinations and no human subjects were involved in the study. Antimicrobial
57
susceptibilities were performed with Vitek2 (Biomèrieux, France) and subsequently confirmed both
58
by broth micro-dilution and Etest. Antibiotics tested were carbapenems (Ertapenem, Meropenem
59
and Imipenem), cephalosporin (Ceftazidime and Cefotaxime), Colistin and Ceftazidime-Avibactam.
60
Carbapenemase activity was first checked by CarbaNP test [9] and after was confirmed by
61
hydrolysis test of raw protein extract. Carbapenemase enzyme type was investigated by the NG-
62
Test Carba 5 (Biotech), an immune-chromatographic assay which use a multiplex Lateral Flow
63
Immune Assay (LFIA) to detect NDM, OXA-48, KPC, VIM and IMP-like and blaKPC gene was
64
amplified by PCR [10] and sequenced (MWG Operons, Germany). ESBL genes (blaCTX-M, blaTEM
65
and blaSHV) were investigated too by PCR [11].
66
Genetic relatedness of the two strains was determined by MLST (Pasteur protocol,
67
https://pubmlst.org/kpneumoniae/) and PFGE.
68
KPC detection by 11,109 Da peak analysis of Maldi-tof spectra was also performed [12-13].
69
Presence of Tn4401 and pO19 elements associated with blaKPC resistance gene have been detected
70
with a PCR reaction [13].
71
PCR Based Replicon Typing (PBRT) protocol [14] was performed to check the plasmid profile of
72
the strains.
73 74
Results
75
Antimicrobial susceptibilities results for both strains are reported in Table 1. The first isolate
76
AMP920, showed meropenem and imipenem resistance, whit MIC of 8 mg/L. The second isolate
77
AMP1009, an isolate later from same patient showed a meropenem MIC of 1 mg/L, while
78
ceftazidime-avibactam MIC between the two isolates change from 1 to 16 mg/L. When AMP920
79
was isolated, since it showed a carbapenem resistant phenotype the strain was checked for
80
carbapenemase activity by CarbaNP test [9] that resulted positive. Carbapenemase enzyme type was
81
investigated by immuno-chromatographic assay (NG-Test CARBA5, NG Biotech) showing the
82
presence of KPC. The second isolate showed an ESBL producer phenotype by Vitek2 system. Since
83
the patient previously presented a K. pneumoniae KPC producer, the second isolate AMP1009 was
84
further investigated. Carbapenem and Ceftazidime-avibactam MICs were checked by Etest first and
85
then confirmed with broth micro-dilution. Also for the AMP1009 isolate CarbaNP and immune-
86
chromatographic test were performed. CarbaNP test for this second isolate resulted negative while
87
the immune-chromatographic test showed the production of KPC enzyme (results summarized in
88
Table 1). Absence or presence of carbapenemase activity was confirmed also by hydrolysis test of
89
raw protein extract and no enzyme able to hydrolyse carbapenems was reported in the AMP1009.
90
To solve this discrepancy, blaKPC gene of both strains were amplified by PCR [10] and sequenced.
91
The sequencing was able to identify a KPC3 variant in the two amplicons. As reported in Figure 1,
92
the blaKPC gene of AMP1009 isolate showed a deletion of 6 nucleotides in position 498-503,
93
bringing a mutate variant with the following deletion of a glutamic acid and leucine in position 167
94
and 168. The complete blaKPC gene sequencing was performed, to investigate genetic differences.
95
The BLAST alignment revealed that blaKPC genes of two isolates matched with the well-known
96
carbapenem-hydrolyzing class A beta-lactamase KPC-3 gene.
97
This mutation affects the proton acceptor active site located in position 167, corresponding to one of
98
the two amino acids deleted. The UniProt analysis of the carbapenem-hydrolyzing β-lactamase KPC
99
(UniProtKB - Q9F663 (BLKPC_KLEPN)), gave us some important informations about where the
100
active sites were located into protein structure and, in detail, about the proton acceptor one. The
101
latter, composed by a glutamic acid (LDRWELELNS), is located at position 167, exactly the site of
102
deletion.
103
BlaSHV, blaTEM were also detected by PCR in both strains.
104
Genetic relatedness of the two strains was determined by MLST (Pasteur protocol) and PFGE. Both
105
strains showed the same MLST profile, corresponding to ST512, belonging to CC256. The clonally
106
of the two isolates was also confirmed by PFGE. The two isolates show a correlation amid 95% and
107
100%, suggesting that those two isolates belong to the same clonal lineage.
108
The peptide mass fingerprint (PMF) generated by MALDI-TOF MS analysis revealed the presence
109
of a 11 109Da peak in both the isolates [10, 11]. This peak is associated to the protein pO19
110
inserted into the same region of the transposon tn4401, often harbouring the blaKPC gene in K.
111
pneumoniae carbapenemase producers' strains. The Tn4401a isoform and pO19 elements associated
112
with blaKPC resistance gene have been detected with a PCR reaction [13].
113
Following the PBRT protocol [14] both the isolates harbour the FIIs plasmid, a pKpQIL-like
114
plasmid often associated with the blaKPC gene.
115
Discussion
116
In our report, we have described a new mechanism of ceftazidime-avibactam resistance with
117
decreasing activity against carbapenems in a K. pneumoniae strain KPC producer in an infected
118
patient. The mechanism of resistance to ceftazidime-avibactam is due to a deletion of 6 nucleotides
119
in position 498-503, that brings to a new variant with a deletion of two amino-acids in position 167-
120
168 The amino-acid in position 167 is involved in the proton acceptor active site.
121
Livermore et al [8] reported the vulnerabilities of ceftazidime-avibactam to select spontaneous
122
mutations bringing to resistance. Winkler et al. [15] reported the activity of ceftazidime-avibactam
123
against a set of isogenic E. coli strains with mutation in the omega-loop of KPC enzyme,
124
demonstrating that substitution of the amino acid positions 165 through 179, enhanced ceftazidime
125
affinity preventing avibactam binding. Our clinical isolate with a deletion of amino acid 167-168 of
126
KPC-3 variant in K. pneumoniae seems to confirm in vivo the importance of mutations in this site to
127
confer ceftazidime-avibactam resistance.
128
An important limitation of this study is the lack of information about patient treatment. Our data
129
support the hypothesis that this type of mutation could be selected by therapy. Now we are planning
130
to investigate on our ceftazidime-avibactam resistant strains and the ceftazidime-avibactam therapy.
131
Another limitation is the lack of characterization of biochemical property such the hydrolytic
132
activity of this variant compare with the KPC-3, even if we know that KPC-3 variant harbored in
133
FII-like plasmid in strains isolated in our hospital setting [ 16] has a size of 97 Kb determined by
134
S1-PFGE and trans conjugant E. coli JC53 showed a meropenem MIC of 1 mg/L.
135
Our report underline also the difficulties of clinical laboratories to reveal these ceftazidime-
136
avibactam resistant isolates since most of them, like in this case were reported as meropenem
137
susceptible, ESBL producing and not screened and identified as KPC producing strain. Also
138
molecular blaKPC detection or immune-chromatographic KPC screening are not able to reveal KPC
139
variants with different affinities and hydrolytic properties.
140
In order to preserve ceftazidime-avibactam activity and monitor its resistance we think that is
141
necessary to test this antibiotic combination not only in presence of a class A carbapenemase but for
142
all strains that present an ESBL phenotype. This assume more importance when patients are known
143
previously colonized or infected with carbapenemase producing Enterobacteria and the new isolates
144
show a carbapenems susceptibility since this may hide a ceftazidime-avibactam resistance.
145 146
Authors’ contribution
147
Elisa Antinori: Investigation, Formal analysis, writing original draft
148
Ilaria Unali; Investigation, validation
149
Anna Bertoncelli: Resources, validation
150
Annarita Mazzariol: Conceptualization, Data curation, writing review-editing, supervision
151
Funding: No external funding was received
152
Conflict of interests: None
153
Acknowledgement: Results were partially presented at the 29th ECCMID, took place in
154
Amsterdam Netherlands, 13-16 April 2019
155 156
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3. Giani T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R et al. Epidemic diffusion of
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That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That
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Function as Extended-Spectrum beta-lactamases. Antimicrob Agents Chemother. 2017:6:
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KPC-3 carbapenemase. Antimicrob. Agents Chemother. 2015.59:5324 –5330.
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9. Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase producing
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molecular technique analysis of a KPC-3-producing Klebsiella pneumoniae outbreak in an
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Italian surgery unit. J.Chemother». 2012:24:93-6.
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11. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex
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Enterobacteriaceae. J Antimicrob Chemother 2010: 65: 490–495.
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12. Lau AF, Wang H, Weingarten RA, Drake SK, Suffredini AF et al. A Rapid MatrixAssisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method
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for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant
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15. Winkler ML, Papp-Wallace KM, Bonomo RA. Activity of ceftazidime/avibactam against
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isogenic strains of Escherichia coli containing KPC and SHV beta-lactamases with single
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amino acid substitutions in the Omega-loop. J Antimicrob Chemother 2015. 70:2279
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16. Kocsis E., Lo Cascio G, Piccoli M, Cornaglia G, Mazzariol A. KPC-3 Carbapenemase
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Harbored in FIIk Plasmid from Klebsiella pneumoniae ST512 and Escherichia coli ST43
205
in the Same Patient. Microb Drug Res. 2014:20:377-82
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Table 1: Antimicrobial susceptibilities and carbapenemase phenotypic tests of the two strains under study.
Strains CZA AMP920 1 AMP1009 16
MICs mg/L ERT >128
4
IMP 8 0.5
MPM 8 1
CAZ >128 >128
CTX 64 4
COL 0,5 0,5
CarbaNP
NGcarba5
pos neg
KPC KPC
CZA: ceftazidime-avibactam; ERT: Ertapenem; IMP: Imipenem; MPM: Meropenem; CAZ: ceftazidime; CTX: cefotaxime; COL: Colistin
Figure 1: Partial Nucleotide and amino acid alignments of blaKPC3 sequence and blaKPC amplicon of AMP1009 strain showing 6 nucleotides deletion. In bold and in the square are reported amino acids position involved in the KPC3 omega loop.
AMP1009 bla KPC3
…………… GTT CCG TCT GGA CCG CTG ___ ___ GGA GCT GAA CTC CGC CAT CCC AGG CGA TGC GCG CGA ………… | || | | | | || | | | | | | || | | | | | || | | | || | | | | | || | | | | | | | | | | | | | | | | | | …………… GTT CCG TCT GGA CCG CTG GGA GCT GGA GCT GAA CTC CGC CAT CCC AGG CGA TGC GCG CGA ……….
AMP1009
…………..
blaKPC3
…………..
F * F
aa position
…………..
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
R * R
L * L
D * D
R * R
W * W
_
_
E
L
E * E
L * L
N * N
S * S
A * A
I * I
P * P
G * G
D * D
A * A
R * R
D * D
…………. …………. ………….