Resistance to ceftazidime–avibactam and other new β-lactams in Pseudomonas aeruginosa clinical isolates: a multi-center surveillance study

ABSTRACT New β-lactam–β-lactamase inhibitor combinations represent last-resort antibiotics to treat infections caused by multidrug-resistant Pseudomonas aeruginosa. Carbapenemase gene acquisition can limit their spectrum of activity, and reports of resistance toward these new molecules are increasing. In this multi-center study, we evaluated the prevalence of resistance to ceftazidime–avibactam (CZA) and comparators among P. aeruginosa clinical isolates from bloodstream infections, hospital-acquired or ventilator-associated pneumonia, and urinary tract infections, circulating in Southern Italy. We also investigated the clonality and content of relevant β-lactam resistance mechanisms of CZA-resistant (CZAR) isolates. A total of 120 P. aeruginosa isolates were collected. CZA was among the most active β-lactams, retaining susceptibility in the 81.7% of cases, preceded by cefiderocol (95.8%) and followed by ceftolozane–tazobactam (79.2%), meropenem–vaborbactam (76.1%), imipenem–relebactam (75%), and aztreonam (69.6%). Among non-β-lactams, colistin and amikacin were active against 100% and 85.8% of isolates respectively. In CZAR strains subjected to whole-genome sequencing (n = 18), resistance was mainly due to the expression of metallo-β-lactamases (66.6% VIM-type and 5.5% FIM-1), followed by PER-1 (16.6%) and GES-1 (5.5%) extended-spectrum β-lactamases, mostly carried by international high-risk clones (ST111 and ST235). Of note, two strains producing the PER-1 enzyme were resistant to all β-lactams, including cefiderocol. In conclusion, the CZA resistance rate among P. aeruginosa clinical isolates in Southern Italy remained low. CZAR isolates were mostly metallo-β-lactamases producers and belonging to ST111 and ST253 epidemic clones. It is important to implement robust surveillance systems to monitor emergence of new resistance mechanisms and to limit the spread of P. aeruginosa high-risk clones. IMPORTANCE Multidrug-resistant Pseudomonas aeruginosa infections are a growing threat due to the limited therapeutic options available. Ceftazidime–avibactam (CZA) is among the last-resort antibiotics for the treatment of difficult-to-treat P. aeruginosa infections, although resistance due to the acquisition of transferable β-lactamase genes is increasing. With this work, we report that CZA represents a highly active antipseudomonal β-lactam compound (after cefiderocol), and that metallo-β-lactamases (VIM-type) and extended-spectrum β-lactamases (GES and PER-type) production is the major factor underlying CZA resistance in isolates from Southern Italian hospitals. In addition, we reported that such resistance mechanisms were mainly carried by the international high-risk clones ST111 and ST235.

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Reviewer #1 (Comments for the Author): This study investigated the prevalence of resistance to CZA and comparators among P. aeruginosa clinical isolates in the context of a regional surveillance program.The topic is of interest, considering the potential use of CZA against MDR P. aeruginosa.Although most issues previously raised by Reviewers were sufficiently addressed, several shortcomings persist.

Major comments:
-the Introduction section is dramatically overlong (e.g.l.67-93), and the manuscript could certainly benefit from a shortened version of this section -Characterization of GES-1-producing strain must be carefully revised.Authors claim a strong promoter (PcS) located upstream of the blaGES-1 gene could explain the observed CZA-R phenotype.However, sequence analysis was significantly flawed, since it cannot confirm the localization of blaGES-1 in a class 1 integron.Indeed, a BLAST analysis on PSA9 clearly showed intI1 and blaGES-1 laying in different contigs.Still, the intI1 sequence was in two different contigs.On top of this, please note that the sequence of the integron cassette promoter (Pc) in PSA9 was not that of a strong promoter, as currently reported in the text.Ref. 42 reported a PcS with a [-35]TTGACA....TAAACT [-10] sequence, but a [-35]TgGACA....TAAACT[-10] sequence was detectable instead, that is a Pc hybrid 1 promoter.Pc hybrid 1 was shown to drive a two-fold increase of blaGES transcription level, roughly (see doi:10.1128/AAC.00912-08).Please modify the text (methods, results) and referencing accordingly.I would also check for the presence of a P2 promoter.
-(not mandatory) I would suggest to partially edit the title, as it appears too declamatory and anticipate somewhat expected results (MBL-mediated CZA resistance) that may inadvertently limit the readers' interest.E.g: "Resistance to ceftazidimeavibactam and other novel β-lactams in Pseudomonas aeruginosa clinical isolates: a multi-center surveillance study" Specific comments: -l.83."generally found in the context of class 1 integrons", I would specify "in Pseudomonads" -l.103 DTR phenotype?-l.231:I would specify 18/22 CZA-R isolates -error values for mean (SD) and median (IQR) should be provided (SNPs analysis) -l.263: if overexpression, than should be blaGES-1 (vs overproduction related to the enzyme, GES-1) Reviewer #2 (Comments for the Author): In the present study, it was carried out the characterization of CZA resistant P. aeruginosa isolates recovered in 5 hospitals in Italy.Although not original, the results are relevant as they contribute to the knowledge in this critical pathogen, considering the few therapeutic options available for the treatment of infections caused by MDR strains.These data also contribute to the epidemiology of the region, in particular of Apulia.I believe that this work would be of interest to the clinical and scientific community, although some changes are recommended for publication.

Mayor comments
The study is not representative of a surveillance study in the region since it only includes one hospital of the Basilicata region.Therefore, I suggest mentioning in the introduction that the aim of this study was to analyze the mechanisms which confer resistance to CZA, to characterized the resistant isolates (circulating clones), and to contribute to the epidemiology of the region.The selection of isolates subject to WGS is confusing.It is mentioned that CZA-resistant isolates were selected, but 18/22 of them were included.On the other hand, of those CZA-susceptible isolates, there were chosen 8 which presented resistance to at least one beta-lactam, and other sensitive ones.May be isolates within each group were randomly selected?Lines 212 to 214: Analyzing the MIC values (MIC50 and MIC90) presented in Table 1, I consider that the differences between the MIC values for CZA respect those for CAZ or PTZ, are not enough to be highlighted in this paragraph.Even more so considering that the values are not absolute but, for example, >16.I suggest rewriting this paragraph.
Lines 247 to 251: Is there any SNP cutoff value to infer clonal relatedness.In my opinion, the SNPs values between some isolates are large enough to assume that these isolates are NOT "high clonal relatedness".Opposite to that mention in the text for both ST 111 and even more for ST235.See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851282/The "Abstract" as well as the "Importance" section should be written according to the suggested modifications.
Minor comments Line 80: avoid repetition of "Furthermore" Line 138: replace "Isolates from cystic fibrosis or pediatric patients" by "Isolates from patients with cystic fibrosis or pediatrics" Line 163: replace "automatic" by "automated" Line 171: replace "strain" by "bacterial genome" Line 204: replace 51.6% by 51,7% and 15.9% by 15,8% Line 287: add: cefiderocol and cefotolozane /tazobactam.Line 289: it must be added: tested negative for carbapenemase "and ESBL coding genes such as blaPER-1 or blaGES-1" Line 305: according to previous comments, It would be better to refer to the fact that the study contributes to local epidemiology Line 609: replace "originating from" by "recovered from"

Reviewer #1 (Comments for the Author):
This study investigated the prevalence of resistance to CZA and comparators among P. aeruginosa clinical isolates in the context of a regional surveillance program.The topic is of interest, considering the potential use of CZA against MDR P. aeruginosa.Although most issues previously raised by Reviewers were sufficiently addressed, several shortcomings persist.

Major comments:
1.The Introduction section is dramatically overlong (e.g.l.67-93), and the manuscript could certainly benefit from a shortened version of this section.Authors' response: We are grateful for your suggestion.We have reduced the length of the Introduction section accordingly (lines 66-78).
2. Characterization of GES-1-producing strain must be carefully revised.Authors claim a strong promoter (PcS) located upstream of the blaGES-1 gene could explain the observed CZA-R phenotype.However, sequence analysis was significantly flawed, since it cannot confirm the localization of blaGES-1 in a class 1 integron.Indeed, a BLAST analysis on PSA9 clearly showed intI1 and blaGES-1 laying in different contigs.Still, the intI1 sequence was in two different contigs.On top of this, please note that the sequence of the integron cassette promoter (Pc) in PSA9 was not that of a strong promoter, as currently reported in the text.Ref. 42 reported a PcS with a [-35]TTGACA....TAAACT [-10] sequence, but a [-35]TgGACA....TAAACT [-10] sequence was detectable instead, that is a Pc hybrid 1 promoter.Pc hybrid 1 was shown to drive a twofold increase of blaGES transcription level, roughly (see doi:10.1128/AAC.00912-08).Please modify the text (methods, results) and referencing accordingly.I would also check for the presence of a P2 promoter.Authors' response: We are grateful to Reviewer 1 for raising this point and for the careful revision.As correctly underscored by Reviewer 1, in the PSA9 draft genome (Illumina sequencing technology), intI1 and blaGES-1 were in different contigs.Therefore, we decided to perform Nanopore sequencing and we carried out a hybrid assembly of the P. aeruginosa PSA9 genome (an update of the new de novo assembly has been deposited at NCBI and is now available with the accession number CP150132).The genome is now closed (the chromosome and the plasmid) and we can now certainly state that blaGES-1 gene is integrated in the chromosome and is located immediately downstream of an incI1 integron carrying a strong promoter (i.e., PcS), which has been previously associated with the overexpression of blaGES-1 (Li et al., 2023(Li et al., doi: 10.1016(Li et al., /j.drup.2023.100973).100973),thus explaining the high CZA MIC observed with this strain (see lines 252-260).We have modified the methods section adding information about Nanopore sequencing and hybrid assembly performed for the PSA9 strain (see lines 175-186).
somewhat expected results (MBL-mediated CZA resistance) that may inadvertently limit the readers' interest.E.g: "Resistance to ceftazidime-avibactam and other novel β-lactams in Pseudomonas aeruginosa clinical isolates: a multi-center surveillance study".Authors' response: We agree with the Reviewer's suggestion.We modified the title accordingly.

Reviewer #2 (Comments for the Author):
In the present study, it was carried out the characterization of CZA resistant P. aeruginosa isolates recovered in 5 hospitals in Italy.Although not original, the results are relevant as they contribute to the knowledge in this critical pathogen, considering the few therapeutic options available for the treatment of infections caused by MDR strains.These data also contribute to the epidemiology of the region, in particular of Apulia.I believe that this work would be of interest to the clinical and scientific community, although some changes are recommended for publication.

Mayor comments
The study is not representative of a surveillance study in the region since it only includes one hospital of the Basilicata region.Therefore, I suggest mentioning in the introduction that the aim of this study was to analyze the mechanisms which confer resistance to CZA, to characterized the resistant isolates (circulating clones), and to contribute to the epidemiology of the region.Authors' response: Thanks for your comment.We have modified the text accordingly (see lines 111-114).
The selection of isolates subject to WGS is confusing.It is mentioned that CZA-resistant isolates were selected, but 18/22 of them were included.On the other hand, of those CZA-susceptible isolates, there were chosen 8 which presented resistance to at least one beta-lactam, and other sensitive ones.May be isolates within each group were randomly selected?Authors' response: We are grateful to reviewer 2 for the careful revision.Unfortunately, 4/22 CZA-resistant strains were not sequenced since they were not culturable after -80°C storage.Thus, only 18 strains were included in the WGS collection.Regarding CZA-susceptible strains, we decided to select strains susceptible to CZA and resistant to ceftolozane/tazobactam and/or at least one carbapenem (including the carbapenem-lactamase inhibitor combinations); unfortunately, also in this category, 7 strains weren't viable after storage and thawing.Finally, only the strains with a non-resistant pattern were selected randomly.We have modified the text to better explain the selection criteria of isolates subjected to WGS (see lines 144-149).
Lines 212 to 214: Analyzing the MIC values (MIC50 and MIC90) presented in Table 1, I consider that the differences between the MIC values for CZA respect those for CAZ or PTZ, are not enough to be highlighted in this paragraph.Even more so considering that the values are not absolute but, for example, >16.I suggest rewriting this paragraph.Authors' response: We agree with the Reviewer 2 and decided to delete this paragraph.
Lines 247 to 251: Is there any SNP cutoff value to infer clonal relatedness.In my opinion, the SNPs values between some isolates are large enough to assume that these isolates are NOT "high clonal relatedness".Opposite to that mention in the text for both ST 111 and even more for ST235.See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851282/

Table 2 :
Replace "Transferable resistance genes" by "Transferable Beta-lactamase coding genes" Supplementary data: Delete table 1 as antibiotic abrevations are included in the text and it is not clear what do you refer with the column "MIC ranges".