Pathogenicity of nosocomial Pseudomonas aeruginosa analyzed by pan-genomics

doi:10.21203/rs.3.rs-3220624/v1

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Pathogenicity of nosocomial Pseudomonas aeruginosa analyzed by pan-genomics

https://doi.org/10.21203/rs.3.rs-3220624/v1

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published 09 Feb, 2024

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Pseudomonas aeruginosa is an important hospital-acquired pathogen that is usually resistant to multiple antibiotics. In 2016–2021, among the 103 strains of P. aeruginosa isolated, the age distribution of patients ranged from 10 days to 94 years with a mean age of 66.38 years. The strains isolated were from sputum (72 strains, 69.9%) and blood (14 strains, 13.6%). The size of these genomes ranged from 6.2 Mb to 7.4 Mb, with a mean value of 6.5 Mb. Besides, 20 antibiotics were selected for the determination of the drug sensitivity test. The results of antibiotic sensitivity tests showed that the isolates had the highest resistance rate of 100% to amoxicillin-clavulanate, ampicillin, ampicillin-sulbactam, cefazolin, cefotaxime, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. In addition to this, a total of 47 crpP genes that mediate resistance to aminoglycoside antibiotics were found distributed on 43 P. aeruginosa strains, and 10 new variants of CrpP were identified, named 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41 and 7.1. Whole genome analysis showed that a total of 50 ST types were identified, with ST244 (5/103) and ST1076 (4/103) having a more pronounced distribution advantage. Serotype predictions showed that O6 accounted for 29.13% (30/103), O11 for 23.30% (24/103), O2 for 18.45% (19/103), and O1 for 11.65% (12/103) of the highest proportions. Notably, we found a significantly higher proportion of ExoU in P. aeruginosa strains of serotype O11 than in other Exo-toxins strains.

Our study enriches the genomic analysis of other multi-drug resistant P. aeruginosa strains, as well as the study of P. aeruginosa carrying CrpP and its variants.

P. aeruginosa

MDR

pan-genome analyse

Serotypes

CrpP

Pseudomonas aeruginosa is a Gram-negative, opportunistic human pathogen that is considered one of the major pathogens associated with hospital-acquired infections, infecting CF lungs and promoting an accelerated decline in lung function [1].

The threat of morbidity and mortality from hospital-acquired infections caused by multidrug-resistant (MDR) or extensively-drug resistant (XDR) strains of P. aeruginosa has increased significantly [2]. P. aeruginosa is characterized by its ability to survive in a wide range of environments, which is closely related to its large genome. The genome size of P. aeruginosa usually ranges from 5.5 Mb to 7 Mb [3]. Dispensable genomes typically consist of horizontally transferable elements, which include integrative and conjugative elements (ICEs), genomic islands (GIs), prophages, transposons, insertion sequences (ISs), and integrons [3].

Quinolones, especially fluoroquinolones (e.g. ciprofloxacin and levofloxacin), are commonly used to treat P. aeruginosa infections. Bacterial resistance to fluoroquinolones is mainly due to reduced outer membrane permeability, overexpression of efflux pumps and/or amino acid substitutions in the sequence of chromosomally encoded topoisomerases that are targets of these antibiotics, as well as the production of plasmid-mediated quinolone resistance (PMQR) genes [4]. Ciprofloxacin (CIP), a fluoroquinolone, is a quinolone that shows considerable activity outside the urinary tract [5]. CrpP, an enzyme capable of phosphorylating ciprofloxacin, was described in 2018 as encoded in plasmid pUM505 from P. aeruginosa; cloning into J53 increased the MIC to ciprofloxacin from 0.008 mg/L to 0.06 mg/L. Since then, crpP-like genes have been reported in E. coli, K. pneumoniae and P. aeruginosa [6–12].

P. aeruginosa has multiple ST types and the top 10 global high-risk clones for P. aeruginosa based on their prevalence, global transmission and association with MDR/XDR profiles include ST235, ST111, ST233, ST244, ST357, ST308, ST175, ST277, ST654 and ST298 [2, 13]. In a study on P. aeruginosa bacteraemia conducted in Spain in 2008–2009, 73 of 81 XDR isolates (90%) were found to belong to only 3 clones, which were the main international MDR/XDR high-risk strains: ST175, ST111 and ST235 [14–17]. Some studies have shown that isolates belonging to serotypes O1, O6, O11 and O12 accounted for more than 65% of P. aeruginosa infections [18, 19]. Serotype O4 and O12 isolates are more often associated with resistance to various antibiotics [20, 21]. P. aeruginosa isolates of serotypes O5, O6 and O11 are commonly found in burn wound infections [22]. O6 and O11 are the most common serotypes in pneumonia [23–25].

In this research, we studied 103 strains of P. aeruginosa isolated from Jiangsu Province from 2016–2021 for resistance phenotypes, antimicrobial resistance genes, pan-genome analyse, ST types and serotypes, and emphasize the diversity of CrpP protein and integration and conjugation elements (ICE) carrying CrpP protein.

2.1 Bacterial strains, media, and culture conditions

In 2016–2021, 103 strains of P. aeruginosa were isolated from patients in various hospitals in Jiangsu Province, China. P. aeruginosa was grown in Luria-Bertani (LB) medium at 37°C. ATCC27853 was used as the control strain for the drug sensitivity test and the recipient strain for the conjugation transfer test.

2.2 Antimicrobial susceptibility of P. aeruginosa isolates

20 antibiotics were selected including Amikacin, Amoxicillin-Clavulanate, Ampicillin, Ampicillin-Sulbactam, Aztreonam, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Chloramphenicol, Ciprofloxacin, Gentamicin, Imipenem, Levofloxacin, Meropenem, Moxifloxacin, Piperacillin, Piperacillin-Tazobactam, Tetracycline, Trimethoprim-Sulfamethoxazole were tested by the agar dilution method and results were determined according to the Clinical and Laboratory Standards Institute (CLSI, 2019) guidelines.

2.3 Whole genome sequencing and analysis

103 strains of P. aeruginosa were sequenced in draft genome by Illumina NovaSeq PE150. CLC Genomics WorkBench software version 22.0 was used for the sequence assembly. Some strains are selected to use Oxford Nanopore Technology for third -generation sequencing. The bacterial genome is annotated by Prokka (v1.14.6) (https://github.com/tseemann/prokka) [26]. ABRicate (https://github.com/tseemann/abricate) is used for the prediction of drug resistance genes and virluence genes. Transposon analysis was predicted on the ICEfinder (https://bioinfo-mml.sjtu.edu.cn/ICEfinder/index.php) website, oriTfinder (https://tool-mml.sjtu.edu.cn/oriTfinder/oriTfinder.html) [27] were used to predict oriT and relaxase on transposon, and transposon gene clusters were mapped at GBKviz (https://moshi4-gbkviz-srcgbkvizgbkviz-webapp-vaurf6.streamlit.app/) and Inkscape. The evolutionary tree of CrpP amino acids was performed using MEGA X, with bootstrap set to 1000. Amino acid alignment of CrpP amino acids sequences was performed using Jalview [28]. MLST (https://github.com/tseemann/mlst) was used to predict the ST type of P. aeruginosa isolates [29]. Phyloviz was used to perform the minimum spanning tree [30]. Pasty (https://github.com/rpetit3/pasty) is used for serotype prediction of P. aeruginosa [31, 32]. Pan-genomic analysis of the gff files generated from Prokka annotations was performed using Roary (https://github.com/ggh2020/Roary) [33]. The newick generated by the Roary annotation was used to generate the evolutionary tree at the chiplot (https://www.chiplot.online/) website. The size of the genome was statistically processed using seqkit (https://github.com/shenwei356/seqkit) [34]. Genetic distances between genomes were calculated by mash (https://github.com/marbl/Mash) [35] .

2.4 Conjugation transfer experiment

Six strains of P. aeruginosa carrying new CrpP variants and MIC ≥ 1 were selected: PA002 (CrpP1.33, MIC(cip) = 1), PA201819 (CrpP1.40, MIC(cip) > 2), PA201616 (CrpP1.36, MIC(cip) > 2), PA202110 (CrpP1.39, MIC(cip) > 2), MIC(cip) > 2), PA201703 (CrpP7.1, MIC(cip) = 1), as donor bacteria for the conjugation test. ATCC27853 (rifampicin-resistant) was selected as the recipient bacteria. The six donor and recipient strains in logarithmic growth phase were mixed in a ratio of 1:3 and incubated on dual anti-MHII plates (1 µg/ml ciprofloxacin, and 50 µg/ml rifampicin) overnight for 12 h. Single colonies of the strains growing on the dual resistance plates were picked and identified by PCR amplification of the crpp gene.

3.1 Bacterial source

Of the 103 P. aeruginosa strains isolated, the distribution of patient age ranged from 10 days to 94 years, with a mean age of 66.38 years. The isolates were sputum (n = 72, 69.9%), blood (n = 14, 13.6%). Table 1 shows the distribution of the sources of the isolates and Affiliation 1 provides all information on the isolates.

Table 1

Distribution of sources of isolated strains
Samples	Number
sputum	72
blood	14
fester	3
secreta	3
urine	3
sanies	2
bile	1
drainage liquid	1
faeces	1
hydrothorax	1

3.2 Results of bacterial drug sensitivity tests

The 101 P. aeruginosa strains were most resistant to Amoxicillin-Clavulanate, Ampicillin, Ampicillin-Sulbactam, Cefazolin, Cefotaxime, Chloramphenicol, Tetracycline, Trimethoprim-Sulfamethoxazole, all with a resistance rate of 100%. The sensitivity rates for Amikacin, Gentamicin, Ceftazidime, Piperacillin, Piperacillin-Tazobactam, Ciprofloxacin, Meropenem, Aztreonam, Imipenem, Cefepime and Levofloxacin were 95.15%, 86.41%, 72.82%, 71.84%, 69.90%, 55.34%, 52.43%, 50.49%, 50.49%, 49.51% and 47.57% respectively (Fig. 1 ). The data from the drug sensitivity tests are listed in Affiliation 2.

3.3 Antimicrobial resistance genes, ST types, serotypes and evolutionary trees

In addition to crpP and its variants, many other important drug resistance genes have been identified through abricate. A total of 61 resistance genes were identified in 103 P. aeruginosa strains. The top five resistance genes were aph(3')-IIb (103, 103/103), fosA (103, 103/103), catB7 (102, 102/103), bla_PDC−374 (77, 77/103), and crpP (47,47/103). Other PMQR resistance genes belonging to the quinolone class, qnrB52 and qnrVC6, were also predicted in the genome of 103 P. aeruginosa strains. In addition, notable carbapenemase-producing resistance genes such as bla_GES−1, bla_GES−14, and bla_VIM−2 were also predicted (Fig. 2).

ST244 (5/103) was found in four separate years, except for the ST type which could not be identified. A total of 50 ST types were identified, and overall these 103 clinical P. aeruginosa isolates had a wide distribution of ST sequences. Minimum spanning tree plotting using phyloviz (Fig. 3).

3.4 Classification, number and amino acid sequence of variants of CrpP

A total of 47 crpP genes, in 43 P. aeruginosa strains, were predicted after abricate prediction analysis. Reference to the classification according to Zhichen Zhu [36]. In total, 17 CrpP variants were identified, including nine new variants of 1.1, named 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, and one CrpP variant with a higher degree of variation, named 7.1.

With the exception of CrpP1.1, the amino acid ranked seventh is the one with the highest frequency of variants, with (7/47) mutating to H and (39/47) amino acids mutating to D. The second most frequent mutation is in the fourth position, K, mutated to R, in this 24 CrpP amino acid sequence. Compared to CrpP1.1, CrpP7.1 has a mutation in the first five amino acids, which is where it differs most from other CrpP1.1 amino acids (Fig. 4).

3.5 Comparison of transposon gene clusters carrying novel variants of CrpP

PA201607, PA201613, PA201614, PA201616, PA201727, PA202110, PA201819, PA201703, PA201820 carry transposons Tn7578-Tn7586 respectively. Among the 9 variants mentioned above, Tn7586 (PA201820) has a total length of 349 kb and differs greatly from the other transposons. In addition to carrying the crpP gene, it also carries the tet(A), msr(E), mph(E), qnrVC6, aac(6')-IIa, catB11, dfrA1, and sul1 genes. Tn7585 carries variant 7.1 with a nucleotide length of 179 kb, second in length only to Tn7586 (Fig. 5).

3.6 The result of Conjugation transfer experiment

Neither the donor nor the recipient mixture grew single colonies on the dual resistance plates, indicating that the transposon carrying crpp failed to transfer into ATCC27853 by conjugation.

3.7 Carriage of the exotoxins in the type III secretion system

The ExoT, ExoY, ExoS and ExoU proteins of the type III secretion system were the focus of attention. The proportion of ExoU in 103 P. aeruginosa was 24.27% (25/103), ExoS 76.70% (79/103), ExoY 96.17% (99/103), and ExoT 100% (103/103). The percentages of ExoU (9/43), ExoS 76.74% (33/43), ExoY 97.67% (42/43), and ExoT 100% (43/43) among the 43 crpp positive P. aeruginosa, respectively. The proportion of O11 in the ExoU, ExoS, ExoY and ExoT P. aeruginosa was 50%, 7.59%, 24.24% and 23.30% respectively. The proportion of ExoU in serotype O11 was significantly higher than that of other strains (Fig. 6).

3.8 Pan-genome analysis

The main reference strain for genetic and functional studies of P. aeruginosa, PAO1 (ACCESSION: NC_002516), the "Liverpool endemic strain" P. aeruginosa LESB58 (NC_011770), which was found to be highly transmissible in CF patients, the ExoU-positive strain PA14 (NC_008463), the Argentine clinical isolate PA7 (NC_009656), which was reported to have an unusual antibiotic resistance pattern, which was reported to have an unusual antibiotic resistance pattern, were selected as reference strains with different characteristics for clustering analysis with the isolates (Fig. 7) [37].

The sizes of these genomes ranged from 6,195,834 bp to 7,353,881 bp, with a mean value of 6,521,728 bp, and a total of seven sequences larger than 7 Mb. A core genome alignment was generated with Roary, the total number of genes in the 103 P. aeruginosa is 18,913. 4,962 core genes (26.24% of all isolates), soft core genes (1.18% of all isolates) comprising 224 genes, 1,169 shell genes (6.18% of all isolates) and 12,558 cloud genes (66.40% of all isolates) were identified (Fig. 8).

A Mash distance of 0 means that the genomes of the bacteria are identical, a Mash distance of 1 means that the genomes of the k-mers are not shared, and a Mash distance less than or equal to 0.5 is approximately equal to 95% average nucleotide identity (ANI) and 70% DNA-DNA hybridization (Fig. 9).

3.9 Genome Accession Numbers and Transposon numbers

BioProject ID number submitted on NCBI is PRJNA945332. The sequences carrying the newly identified CrpP variant transposons in this study were registered on the transposon registry (https://transposon.lstmed.ac.uk/), generating the accession numbers Tn7578 - Tn7586 [34] .

In this study, 101 human-derived P. aeruginosa strains isolated from Jiangsu Province, China, were subjected to bacterial resistance studies and genome-wide analyses including ST types, serotypes and resistance genes, with a focus on the classification of CrpP variants and mobile elements carrying CrpP variants. The sensitivity to Amoxicillin-Clavulanate, Ampicillin, Ampicillin-Sulbactam, Cefazolin, Cefotaxime, Chloramphenicol, Moxifloxacin, Tetracycline and Trimethoprim-Sulfamethoxazole was 0, which is not recommended for clinical use. Amikacin, Gentamicin, Ceftazidime, Piperacillin and Piperacillin-Tazobactam were recommended with sensitivity rates of 95.15%, 86.41%, 72.82%, 71.84% and 69.90% respectively. The sensitivity rate for Ciprofloxacin was 55.34%. In 2015, the European Centre for Disease Prevention and Control stated that 13.7% of P. aeruginosa isolates were resistant to at least three major classes of antibiotics. In terms of antimicrobial resistance phenotypes, all of the P. aeruginosa strains isolated from patients in this study were MDR strains, accounting for 100% of the total.

Meanwhile, the analysis of ST research shows that with the exception of the unidentifiable ST type, ST244 (5/103) was found in all four years respectively. The next most frequent ST type was ST1076 (4/103). Overall, these 103 clinical P. aeruginosa isolates had a wide variety of ST types, with a total of 50 ST types identified. A study of 100 strains of P. aeruginosa isolated in France and Switzerland between 2000 and 2015 showed that the most prevalent types of ST for crpP-positive (46/100) isolates were ST235, ST111, ST233 and ST273 (24%, 17.4%, 6.5% and 4.3% respectively) [38]. The ST types prevalent in this study are completely different from the main prevalent ST types of crpP-positive P. aeruginosa in European countries, presumably related to regional differences, as well as variability between strains. ST244 has also been reported to be frequently detected in several countries, but not always in association with MDR/XDR strains [39, 40].

Between 2005–2017, the most prevalent serotypes of P. aeruginosa found in 413 patients from 10 countries on four continents were O11 (n = 89; 22%), O1 (n = 58; 14%) and O6 (n = 53; 13%) [25]. A survey of 1445 P. aeruginosa strains conducted in Spain in 2017 found that the most common serotypes were O6 (17.8%), O1 (15.4%) and O11 (13.3%) [21]. Our study found that serotypes included O6, O11, O2 and O1, accounting for 29.13% (30/103), 23.30% (24/103), 18.45% (19/103) and 11.65% (12/103) respectively, these four serotypes together account for more than 80% of the total, and do not differ significantly from the prevalent serotypes of human origin in other countries.

ExoU has been reported to significantly enhance the virulence of P. aeruginosa [41]. Among the various phenotypes of P. aeruginosa isolates, clinical isolates of serotype O11 were found to secrete ExoU, more frequently than other serotypes and serotype O11 was associated with increased lung injury in a mouse model of pneumonia [42]. It is note worthy that our study shows the O11 serotype accounts for 50% of the ExoU positive strains, which is significantly higher than the percentage of ExoS, ExoY and ExoT positive P. aeruginosa strains. Most P. aeruginosa strains secrete either ExoS or ExoU, but not both. It is rare for a single strain of bacteria to carry both ExoU and ExoS. In our collection of 103 human-derived P. aeruginosa strains, only one strain was present with both ExoU and ExoS [43].

In the analysis of antimicrobial resistance gene, aph(3')-IIb (103, 103/103), fosA (103, 103/103), and catB7 (102, 102/103), which mediated resistance to aminoglycosides, fosfomycin, and chloramphenicol, respectively, and were found most frequently. Nevertheless, the first-line drug resistance genes of interest, such as tet(X), which mediates tigecycline, and mcr-1, which mediates polymyxin, have not been identified. Quinolones are considered to be common agents in the treatment of P. aeruginosa infections. Among the PMQR genes that mediate resistance to quinolones, qnrB52 (PA201944), and qnrVC6 (PA201820), were also predicted to present resistance to ciprofloxacin as resistant and mediated, respectively. Plasmid-mediated quinolone resistance (PMQR) genes, including the Qnr protein, which prevents quinolone binding to target proteins, have been reported to reduce susceptibility to ciprofloxacin [44].

Another gene of interest, crpp remains controversial as it is presumed to be transferable and resistant to ciprofloxacin. In the analysis of resistance genes, crpp was found to have many nucleotide mutations, and then its amino acid mutant variants continued to be investigated and classified based on the article published by Zhichen Zhu [36]. Nine new derivative variants of CrpP1.1 were identified, named 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41 and one variant of CrpP with a higher degree of variability, named 7.1. In addition to this, transposons encoding both CrpP were identified in four strains of P. aeruginosa. 47/103 CrpP variants were identified in this study, representing 45.63%. The top five CrpP in terms of number are CrpP1.18 (10/47), CrpP1.2 (9/47), CrpP1.31 (4/47), CrpP1.17 (3/47), CrpP1.34 (3/47). In a separate survey of CrpP variants of P. aeruginosa, strains were isolated mainly from patients in eastern China, and a total of 117 CrpP variants were found in 200 P. aeruginosa strains, representing a total of 58.5% [36].

With the exception of CrpP1.1, the amino acid in the seventh position is the amino acid that appears most frequently as a variant, with (7/47) mutating to H and (39/47) amino acids mutating to D. The second most frequent mutation is in the fourth position, K, which mutates to R and occurs in 24 CrpP amino acids. Compared to CrpP1.1, CrpP7.1 has a mutation in the first five amino acids, which is where it differs most from CrpP1.1. The CrpP protein has 66 amino acids and is more conserved in the N-terminal region, with only amino acids 1.20 and 1.40 mutated to K at position 62, presumably with residues important to its function in the N-terminal region. However, our experimental studies have shown that the CrpP variant cannot be transferred into the recipient bacterium ATCC27853 by conjugational transfer, suggesting that transposons carrying CrpP do not have the ability to transfer naturally. Meanwhile, only 10 of the 47 strains carrying the CrpP variant were resistant to ciprofloxacin, carrying variants classified as CrpP1.2, CrpP1.4, CrpP1.8, CrpP1.18, CrpP1.31, CrpP1.36, CrpP1.39. The effect of mutations at different positions of the CrpP amino acid on quinolones is still worth exploring. Besides, one study reported that the exoU gene was positively correlated with the crpP gene [36]. However, our study showed no significant difference in the number of strains carrying the exotoxins between the crpp-positive and non-positive groups.

In this study, 101 strains of P. aeruginosa isolated from China between 2016-2021, were studied for drug resistance, resistance genes, prevalent serotypes, ST types and pan-genome analysis with a focus on the transferability of crpp-carrying variants and the structure of their variants, enriching the epidemiological data on clinical P. aeruginosa.

Ethical Approval and Consent to participate

Not Applicable.

Consent for publication

Everyone agrees to publish this article.

Availability of supporting data

The data sets supporting the results of this article are included within the article and its additional files.

Competing interests

We declare that we have no competing of interest.

Funding

This work was supported by grants from the Sanya Institute of Nanjing Agricultural University (NAUSY-MS12) and Hainan Province Science and Technology Special Fund (ZDYF2022XDNY236).

Authors’ contributions

Zimeng Hu designed the experiments and wrote the article, Lu Zhou was responsible for the antimicrobial test, Xinyu Tao participated in the isolation of the bacteria, Pei Li and Xiangkuan Zheng were responsible for the analysis of the genomic data, and Wei Zhang and Zhongming Tan were responsible for revising the article.

Acknowledgements

Not Applicable.

Authors’ information

Zimeng Hu^a^†: [email protected]

Lu Zhou^b†: [email protected]

Xingyu Tao^a: [email protected]

Pei Li^a:[email protected]

Xiangkuan Zheng^a: [email protected]

Wei Zhang^a*: [email protected]

Zhongming Tan^b*: [email protected]

Corresponding author contact information

Dr.Vet Med. ZHANG Wei
College of Vet. Med.
Nanjing Agricultural University

No.1 Weigang, Xuanwu District
Nanjing City, Jiangsu Province
People's Republic of China

Mobile phone: 86-1381 4076 553
E-mail address:
[email protected]

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No competing interests reported.

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published 09 Feb, 2024

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Editorial decision: Major revision
09 Aug, 2023
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Pathogenicity of nosocomial Pseudomonas aeruginosa analyzed by pan-genomics

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Bacterial strains, media, and culture conditions

2.2 Antimicrobial susceptibility of P. aeruginosa isolates

2.3 Whole genome sequencing and analysis

2.4 Conjugation transfer experiment

3. Results

3.1 Bacterial source

3.2 Results of bacterial drug sensitivity tests

3.3 Antimicrobial resistance genes, ST types, serotypes and evolutionary trees

3.4 Classification, number and amino acid sequence of variants of CrpP

3.5 Comparison of transposon gene clusters carrying novel variants of CrpP

3.6 The result of Conjugation transfer experiment

3.7 Carriage of the exotoxins in the type III secretion system

3.8 Pan-genome analysis

3.9 Genome Accession Numbers and Transposon numbers

4. Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1