β-lactamase-mediated resistance in MDR-Pseudomonas aeruginosa from Qatar

doi:10.21203/rs.3.rs-56029/v2

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β-lactamase-mediated resistance in MDR-Pseudomonas aeruginosa from Qatar

https://doi.org/10.21203/rs.3.rs-56029/v2

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published 01 Nov, 2020

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Background: The distribution of b-lactamase resistance genes in P. aeruginosa is often closely related to the distribution of certain high-risk international clones. We used whole-genome sequencing (WGS) to identify the predominant sequence types (ST) and b-lactamase genes in clinical isolates of multidrug-resistant (MDR)-P. aeruginosa from Qatar

Methods: Microbiological identification and susceptibility tests were performed by automated BD Phoenix system and manual Liofilchem MIC Test Strips.

Results: Among 75 MDR-P. aeruginosa isolates; the largest proportions of susceptibility were to ceftazidime-avibactam (n=36, 48%), followed by ceftolozane-tazobactam (30, 40%), ceftazidime (n=21, 28%) and aztreonam (n=16, 21.3%). Almost all isolates possessed Class C and Class D b-lactamases (n=72, 96% each), while metallo-β-lactamases were detected in 20 (26.7%) isolates. Eight (40%) metallo-β-lactamases producers were susceptible to aztreonam and did not produce any concomitant extended-spectrum b-lactamases. High risk ST235 (n=16, 21.3%), ST357 (n=8, 10.7%), ST389 and ST1284 (6, 8% each) were most frequent. Nearly all ST235 isolates (15/16; 93.8%) were resistant to all tested b-lactams.

Conclusion: MDR-P. aeruginosa isolates from Qatar are highly resistant to antipseudomonal b-lactams. High-risk STs are predominant in Qatar and their associated MDR phenotypes are a cause for considerable concern.

Infectious Diseases

General Microbiology

beta-lactamases

MDR

P. aeruginosa

ST235

Due to their established efficacy and safety, anti-pseudomonal b-lactam antibiotics play a vital role in the clinical management of P. aeruginosa infections [1]. Key antimicrobial resistance mechanisms in P. aeruginosa include over-expression of efflux pumps, impermeability through porin modification or loss, target modification, and enzyme-mediated antimicrobial inactivation (e.g., b-lactamases). Multiple resistance mechanisms are frequently present in concert resulting in simultaneous resistance to multiple agents [1]. The epidemiology of b-lactamases is often closely related to the distribution of certain high-risk international clones [2]. In this study, we used whole-genome sequencing (WGS) to identify the predominant sequence types (STs) and b-lactamase genes in multi-drug resistant (MDR) P. aeruginosa clinical isolates from Qatar.

The study setting, bacterial identification, antimicrobial susceptibility testing, whole genome sequencing, and statistical analysis details are provided in Additional File 1. MDR status was defined as in-vitro resistance to at least one agent from three or more classes of anti-pseudomonal agents [3]. b-lactamases were classified according to their molecular groups [4]. Clinical data were retrieved from the electronic healthcare system.

Seventy-five MDR-P. aeruginosa isolates were included (Table S1, Additional File 1). The largest proportions of susceptibility were to ceftazidime-avibactam [36, 48%; minimum inhibitory concentration (MIC)_50/90 12/256 µg/ml] and ceftolozane-tazobactam (30, 40%; MIC_50/90 24/256 µg/ml) (Figure 1). Four (5.3%) isolates were resistant to all tested b-lactams except ceftazidime-avibactam, while only one (1.3%) isolate was only susceptible to ceftolozane-tazobactam (Table S1, Additional File 1).

Almost all isolates possessed Class C and Class D b-lactamases (72, 96% each). All 4 b-lactamase classes were present in 3 (4%) isolates. Metallo-b-lactamases (MBL) were detected in 20 (26.7%) isolates. Eight (40%) MBL producers were susceptible to aztreonam and did not produce any concomitant extended-spectrum b-lactamases (ESBL) (Table S1, Additional File 1).

The most frequent STs identified were ST235 (16, 21.3%) and ST357 (8, 10.7%) (Figure 2). All but one ST235 isolate were resistant to all tested b-lactam agents. Furthermore, amongst the 16 ST235 MDR-P. aeruginosa isolates included in this study, MBL were detected in nine (56.3%), bla_VEB-9 in 8 (50%), bla_PDC-2 in 15 (93.8%), and bla_OXA-10 and bla_OXA-50 in all 16 (100%). There were five ST233 MDR-P. aeruginosa isolates; all possessed bla_VIM-2, bla_PDC-3, bla_OXA-4 and bla_OXA-486, and four (80%) of them were resistant to all tested b-lactams except aztreonam. Different patterns of b-lactamase genes and b-lactam susceptibility were observed in other STs (Table S2, Additional File 1).

The present study included MDR-P. aeruginosa isolates from a national diagnostic microbiology laboratory in Qatar and are therefore representative of the whole country. Notably, MDR-P. aeruginosa in Qatar are highly resistant to b-lactam agents. The most active b-lactam antibiotics in this study were those in combination with b-lactamase inhibitors, ceftazidime-avibactam and ceftolozane-tazobactam, were not available for clinical use at the time of the study. Yet, less than half of the isolates were susceptible. Given their recent availability for patients in Qatar, the results reported demonstrate the importance of their appropriate clinical use to minimize further loss of activity [5].

This report included 20 (26.67%) isolates that possessed 21 MBL-encoding genes (16 bla_VIM-2, 2 bla_VIM-5,and 3 bla_IMP-2) (Table S1 and Table 2, Additional File 1). This is consistent with the known predominance of Verona integron-encoded metallo-β-lactamases (VIM), and to a lesser extent imipenemases (IMP), in P. aeruginosa from the Middle East [6-8]. Unlike other geographic settings, New Delhi metallo-b-lactamases (NDM) have not been detected in P. aeruginosa from the Arabian Peninsula [7, 9].

Apart from areas with a high prevalence of MBL in P. aeruginosa, the presence of Class A ESBL b-lactamases can result in resistance to ceftolozane-tazobactam [1]. Avibactam is an efficient inhibitor of Class A b-lactamases and hence ceftazidime-avibactam combination retains its activity in this situation but not ceftolozane-tazobactam [10, 11]. In a report from Spain of 24 extremely-drug resistant ST235 P. aeruginosa isolates, 13% were susceptible to ceftolozane-tazobactam and 58% to ceftazidime-avibactam and the predominant b-lactamases identified were VIM-2 (42%) and the Class A ESBL Guiana-Extended-Spectrum (GES)-5 (46%) [12]. Consistent with this, five out of seven ceftolozane-tazobactam-resistant, ceftazidime-avibactam-susceptible MDR-P. aeruginosa isolates in our study possessed class A bla_SHV-11and ESBL-encoding genes such as bla_VEB-9 and bla_TEM-116. Interestingly, those 7 isolates belonged to seven different STs (Table S1, Additional File 1).

The b-lactamase bla_VEB-9 (19, 25.33%), formerly known as bla_VEB-1a, was the most frequent ESBL gene identified in the present study [11]. bla_VEB-1 is one of the most frequently reported ESBLs in P. aeruginosa from the Middle East including Kuwait, Saudi Arabia and Iran [13-15]. Though bla_VEB-9 was reported from Thailand and Eastern Europe, to the best of our knowledge, it has not been previously reported from the Middle East [11, 16]. In this study, MDR-P. aeruginosa producing Vietnamese extended-spectrum beta-lactamase-9 (VEB-9) belonged to ST235 (8/16), ST357 (7/8), ST308 (1/3) and ST3022 (1/1) (Table S1 and Table S2, Additional File 1). This pattern suggests dissemination within specific P. aeruginosa STs in Qatar that may be different from neighboring countries.

An interesting observation in this study was that 16 (21.33%) MDR-P. aeruginosa isolates were susceptible to aztreonam but resistant to several other antipseudomonal b-lactams tested (Table S1, Additional File 1). Aztreonam is a weak inducer of Class C enzymes and is not a substrate for Class B and narrow-spectrum Class D b-lactamases [17]. The retained aztreonam activity in these isolates despite resistance to other antipseudomonal b-lactams may be explained by the absence of Class A ESBL in those isolates. Therefore, aztreonam should be included in routine antimicrobial susceptibility testing of clinical P. aeruginosa isolates.

Most MDR-P. aeruginosa isolates included in this study belonged to five STs and had consistent b-lactamase genetic profiles and b-lactam susceptibility patterns (Table S2, Additional File 1). ST235, ST233, and ST357 are already known as high-risk clones in Qatar, Saudi Arabia, Bahrain, and the United Arab Emirates [7]. These three STs are globally disseminated MDR-P. aeruginosa clones [2]. Often, these strains cause regional or nationwide outbreaks, express MDR phenotypes, and are associated with high mortality [12, 18, 19]. VIM-producing ST1284 P. aeruginosa have been described from Brazil, and ST389 from cystic fibrosis patients in Italy [20, 21]. Both sequence types have otherwise limited geographic distribution.

MDR-P. aeruginosa isolates from Qatar are highly resistant to antipseudomonal b-lactams. Global high-risk STs predominate in Qatar and their associated multi-resistant phenotype is a cause for considerable concern.

CLSI: Clinical Laboratory Standards Institute; ESBL: extended-spectrum b-lactamase; GES: Guiana-Extended-Spectrum; IMP: imipenemase; MBL: metallo-b-lactamase; MIC: minimum inhibitory concentration; MDR: multi-drug resistant; NDM: New Delhi metallo-b-lactamase; ST: sequence type; VEB: Vietnamese extended-spectrum b-lactamase; VIM: Verona integron-encoded metallo-β-lactamase; WGS: whole-genome sequencing.

Authors’ Contribution

Conception and design of study: M.A.S., E.B.I.; Acquisition of data: M.A.S., F.A.K.; Data analysis and interpretation: M.A.S., A.S.O.; Drafting of manuscript: M.A.S., A.S.O. All authors critically reviewed the manuscript and agreed to its publication.

Funding

The publication is supported by a grant from Qatar National Library. The study was funded by an internal research grant (IRGC-01–51-033 to EI) from the Medical Research Centre at Hamad Medical Corporation, Doha, Qatar. Support was also provided by NPRP grant (NPRP12S-0219-190109 to AAS) from the Qatar National Research Fund (a member of Qatar Foundation) and the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (Formas) (grant 219-2014-837 to JJ and BS). The funders were not involved in the conduct of the study, the preparation of the manuscript, or the decision to submit the manuscript for publication.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval

This study was approved by the Research Ethics Committee (Protocol number IRGC-01-51-033) at Hamad Medical Corporation, Doha, Qatar.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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β-lactamase-mediated resistance in MDR-Pseudomonas aeruginosa from Qatar

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