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Article

In Vitro Activity of Ceftolozane-Tazobactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Obtained from Blood Cultures from Sentinel Public Hospitals in South Africa

by
Olga Perovic
1,2,*,
Ashika Singh-Moodley
1,2 and
Michelle Lowe
1,2
1
Centre for Healthcare-Associated Infections, Antimicrobial Resistance and Mycoses, National Institute for Communicable Diseases a Division of the National Health Laboratory Service, Johannesburg 2192, South Africa
2
Department of Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of Witwatersrand, Johannesburg 2193, South Africa
*
Author to whom correspondence should be addressed.
Antibiotics 2023, 12(3), 453; https://doi.org/10.3390/antibiotics12030453
Submission received: 17 January 2023 / Revised: 16 February 2023 / Accepted: 22 February 2023 / Published: 24 February 2023
(This article belongs to the Special Issue Multi-Drug Resistant Gram-Negative Infections: Molecular Epidemiology, Microbiological Diagnosis and Antimicrobial Treatment)
Versions Notes

Abstract

:
Multidrug-resistant (MDR) Gram-negative bacteria are responsible for the majority of healthcare-associated infections and pose a serious threat as they complicate and prolong clinical care. A novel cephalosporin-β-lactamase-inhibitor combination, ceftolozane-tazobactam (C/T) was introduced in 2014, which improved the treatment of MDR pathogens. This study aimed to evaluate the activity of C/T against Escherichia coli (n = 100), Klebsiella pneumoniae (n = 100), and Pseudomonas aeruginosa (n = 100) blood culture isolates in South Africa (SA). Isolates were sequentially selected (2010 to 2020) from the Group for Enteric, Respiratory, and Meningeal Diseases Surveillance (GERMS) programme in SA. Organism identification was performed using the matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) instrument (Microflex, Bruker Daltonics, Bremen, Germany), and antibiotic susceptibility was performed using the Sensititre instrument (Trek Diagnostic Systems, East Grinstead, UK). C/T resistance was reported in 16 E. coli, 28 K. pneumoniae and 13 P. aeruginosa isolates. Fifty percent of the C/T resistant isolates were subjected to whole-genome sequencing (WGS). According to the whole genome multilocus sequence typing (MLST) analysis, the E. coli isolates (n = 8) belonged to sequence type (ST)10, ST131, ST405, and ST410, the K. pneumoniae isolates (n = 14) belonged to ST1, ST37, ST73, ST101, ST231, ST307, ST336 and ST6065 (novel ST), and the P. aeruginosa isolates (n = 7) belonged to ST111, ST233, ST273, and ST815. The WGS data also showed that all the E. coli isolates harboured aminoglycoside (aph (3′′)-Ib, aph (6)-Id), macrolide (mdfA, mphA), and sulphonamide (sul2) antibiotic resistance genes, all the K. pneumoniae isolates harboured β-lactam (blaCTX-M-15), and sulphonamide (sul2) antibiotic resistance genes, and all the P. aeruginosa isolates harboured aminoglycoside (aph (3′)-IIb), β-lactam (PAO), fosfomycin (fosA), phenicol (catB7), quinolone (crpP), and disinfectant (qacE) antibiotic resistance genes. It is evident that E. coli, K. pneumoniae and P. aeruginosa can adapt pre-existing resistance mechanisms to resist newer β-lactam molecules and inhibitors, since these isolates were not exposed to ceftolozane-tazobactam previously.

1. Introduction

Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are frequently isolated pathogens in the community and healthcare settings, and are the causes of infection-related morbidity and mortality [1,2]. Of concern is that these pathogens are becoming more resistant to available antibiotics [1].
A new cephalosporin-β-lactamase-inhibitor combination, ceftolozane/tazobactam (C/T) (sold under the brand name Zerbaxa, MERCK Connect, United States of America (USA)) was introduced in 2014 and has improved the treatment of carbapenem-resistant Gram-negative pathogens, especially P. aeruginosa [3,4]. Ceftazidime-avibactam (C/A), introduced in 2015 is also a combination antibiotic like C/T and consists of a broad-spectrum cephalosporin (i.e., ceftazidime) and a β-lactamase inhibitor (i.e., avibactam) [5].
C/T is a combination antibiotic that consists of a fifth-generation cephalosporin (ceftolozane) and a β-lactamase inhibitor (tazobactam) that exhibits bactericidal properties [6,7]. Ceftolozane, like other β-lactams, inhibits penicillin binding proteins (PBPs), which prevents bacterial cell wall synthesis and leads to cell death [1. Tazobactam inhibits most class A β-lactamases and some AmpC cephalosporinases (plasmid-mediated class C β-lactamases) and protects ceftolozane from hydrolysis [1,7]. C/T and C/A have been registered drugs in South Africa (SA) since 2022 [8]. Previously, these antibiotics were prescribed in SA as Section 21 drugs (i.e., before these antibiotics can be administered to patients, approval is needed from the hospital’s drug and therapeutic committee/s and the South African Health Products Regulatory Authority (SAHPRA)) [8,9]. These combination antibiotics are costly and not easily accessible in SA [9].
The use of C/T and C/A in SA is predominantly driven by the private healthcare sector [8]. The public healthcare sector in SA does not routinely offer susceptibility testing for C/T and C/A [9]. A comprehensive South African guide for new and existing antibiotics, which includes dose and administration strategies for critically ill patients has recently been published [8]. The focus on appropriate antibiotic stewardship practices is vital to maximise the efficacy and longevity of all new antibiotics that enter the healthcare sector [8].
Monitoring the susceptibility profile of these new combination antibiotics is important as increasing levels of resistance to C/T are observed, putting long-term use at risk [8].
This study aimed to evaluate the in vitro activities of C/T against E. coli, K. pneumoniae and P. aeruginosa and to molecularly characterise selected C/T resistant isolates using whole genome sequencing (WGS).

2. Results

2.1. Demographic Characteristics of Patients

E. coli (n = 100), K. pneumoniae (n = 100), and P. aeruginosa (n = 100) blood culture isolates were collected from 2010 to 2020. The median age for patients with E. coli, K. pneumoniae and P. aeruginosa bacteraemia was 36 years, 31 years and 44 years, respectively. The demographic characteristics of the patients are described in Table 1.

2.2. Phenotypic Characterisation of All Blood Culture Isolates

The antibiotic susceptibility results are shown in Table 2.
The six most active antibiotics against E. coli were C/A (susceptibility rate 96%), meropenem (susceptibility rate 91%), imipenem (susceptibility rate 89%), amikacin (susceptibility rate 89%), gentamicin (susceptibility rate 88%) and C/T (susceptibility rate 82%). Colistin showed intermediate resistance of 94%.
The six most active antibiotics against K. pneumoniae were CA (susceptibility rate 100%), meropenem (susceptibility rate 100%), imipenem (susceptibility rate 99%), amikacin (susceptibility rate 99%) and C/T (susceptibility rate 66%). Colistin showed intermediate resistance of 94%.
The six most active antibiotics against P. aeruginosa were amikacin (susceptibility rate 86%), C/T (susceptibility rate 85%), C/A (susceptibility rate 84%), gentamicin (susceptibility rate 78%), tobramycin (susceptibility rate 78%), and aztreonam (susceptibility rate 76%). Colistin showed intermediate resistance of 68%.
Thirty percent (30/100) of the E. coli, 61% (61/100) of the K. pneumoniae, and 24% (24/100) of the P. aeruginosa isolates were multidrug resistant (MDR).

2.3. WGS of Selected Blood Culture Isolates

Half (50%) of the C/T resistant E. coli (n = 8/17), K. pneumoniae (n = 14/28), and P. aeruginosa (n = 7/13) isolates were subjected to WGS (Table A1).

2.3.1. E. coli Isolates

The average genome size of the sequenced E. coli isolates was 5035 kb with a coverage depth of 68 × to 133 ×. The average G + C content was 50.
The E. coli isolates belonged to four different sequence types (STs) (Table 3). The E. coli isolates harboured between one to seven β-lactamase genes. In total, 138 antibiotic resistance genes were found in the sequenced E. coli isolates. All the E. coli isolates harboured the aph (3′′)-Ib, aph (6)-Id, mdfA, mphA and sul2 genes (Table 3). The majority of the isolates harboured the aac (6′)-Ib-cr, aadA5, blaCTX-M-15, blaOXA-1, drfA17 and sul1. No AmpC-genes (also known as Pseudomonas-derived cephalosporinase (PDC) genes) were detected. All the E. coli isolates harboured the IncFIB (AP001918) and IncFIA plasmids (Table 4).

2.3.2. K. pneumoniae Isolates

The average genome size of the sequenced K. pneumoniae isolates was 5611 kb with a genome coverage of 32× to 125×. The average G + C content was 57.
The K. pneumoniae isolates belonged to eight different STs of which ST6065 is a novel ST (i.e., a newly assigned ST by the BIGSdb-Pasteur database) (Table 3). The K. pneumoniae isolates harboured between one to 14 β-lactamase genes. In total, 312 antibiotic resistance genes were found in the sequenced K. pneumoniae isolates. All K. pneumoniae isolates harboured the blaCTX-M-15, and sul2 genes (Table 3). The majority of the isolates harboured the aph (3′′)-Ib, aph (6)-Id, blaOXA-1, blaTEM-1B, fosA, catB3, OqxA and OqxB genes. No AmpC-genes were detected. The majority of the K. pneumoniae isolates harboured the IncFII (K) (n = 13), IncFib (K) (n = 12) and IncR (n = 11) plasmids (Table 4).

2.3.3. P. aeruginosa Isolates

The average genome size of the sequenced P. aeruginosa isolates was 6974 kb with a genome coverage of 57× to 84×. The average G + C content was 50.
The P. aeruginosa isolates belonged to four different STs (Table 3). The P. aeruginosa isolates harboured between one to seven β-lactamase genes. In total, 99 antibiotic resistance genes were found in the sequenced P. aeruginosa isolates. All P. aeruginosa isolates harboured the aph (3′)-Iib, PAO, fosA, catB7, crpP and qacE genes (Table 3). The majority of the isolates harboured the aadA2, aac (6′)-II, blaVIM-2, dfrB5 and sul2 genes. No AmpC-genes were detected. No plasmids were detected in the sequenced P. aeruginosa isolates (Table 4).
All the sequenced E. coli, K. pneumoniae and P. aeruginosa isolates were resistant to cephalosporins (cefotaxime and ceftazidime) (Table A2). All the E. coli and K. pneumoniae isolates were resistant to aztreonam except for most of the P. aeruginosa isolates, which were shown to be susceptible. All E. coli and P. aeruginosa isolates were resistant to one or more tested carbapenems. However, all the sequenced K. pneumoniae isolates were susceptible to all tested carbapenems.

3. Discussion

With the continuous rise of multidrug resistance in K. pneumoniae and P. aeruginosa, and to a lesser extent in E. coli there are only a few treatment options left for infected patients [7]. With the introduction of C/T in 2014, it has proven to be highly effective against MDR Gram-negative pathogens. The in vitro activity of C/T, against E. coli, K. pneumoniae, and P. aeruginosa isolates obtained from blood cultures from sentinel public hospitals in SA were investigated. The majority of the isolates were collected from adult patients in the Gauteng province; this province has the highest population in South Africa [10].
This study showed that the majority of the E. coli, and P. aeruginosa isolates are highly susceptible to C/T (82% to 85%). In comparison, K. pneumoniae isolates indicated decreased susceptibility towards C/T (66%). Findings from a previous study showed similar C/T susceptibility rates to E. coli, K. pneumoniae, and P. aeruginosa [7]. In contrast, higher C/T susceptibility rates in K. pneumoniae were reported in Germany and the USA [1,11]. The decreased susceptibility to C/T in the K. pneumoniae isolates reported in this study could potentially be due to increased antibiotic resistance [7]. In this study, we have detected multiple β-lactamase genes in the sequenced K. pneumoniae isolates (1 to 14 β-lactamase genes per isolate).
Interesting to note is that all the isolates were highly susceptible to C/A (84% to 100%). The tested E. coli isolates showed ≥80% susceptibility towards amikacin, imipenem and meropenem, K. pneumoniae isolates showed ≥80% susceptibility towards amikacin, ertapenem, imipenem and meropenem, and P. aeruginosa isolates showed ≥80% susceptibility towards amikacin. All E. coli (94%), K. pneumoniae (94%), and P. aeruginosa (68%) isolates showed intermediate resistance to colistin.
In this study, multidrug resistance was detected in 30% of the E. coli isolates, 61% of the K. pneumoniae isolates, and 24% of the P. aeruginosa isolates (Table 2). C/T was not effective against the majority of MDR isolates detected in this study. In contrast, other studies reported that treatment with C/T generally led to favorable clinical outcomes among patients with MDR, extensively drug-resistant (XDR) or pan drug-resistant (PDR) bloodstream infections associated with E. coli, K. pneumoniae, and P. aeruginosa [12,13,14,15].
WGS is a powerful tool that can be used to characterise the genetic diversity of bacterial populations and can also be used for the prediction of bacterial antibiotic resistance profiles [16]. However, the prediction of susceptibility or resistance to antibiotics based only on the presence or absence of previously known genes is still under investigation and discordances are reported in the literature [16]. Half of the C/T resistant isolates were subjected to sequencing for genomic characterisation. AmpC (PDC) and blaGES hyper-production are factors linked to C/T resistance [3,4,17,18,19,20]. However, the blaGES and AmpC genes were not detected in our isolate collection that was subjected to WGS. Other antibiotic resistant genes reported in C/T resistant isolates are the blaCTX-M, and blaSHV genes [18]. The blaCTX-M-15 gene was detected in the majority of the E. coli isolates, and in all the K. pneumoniae isolates. The blaSHV genes were only detected in K. pneumoniae isolates. The precise resistance mechanisms leading to C/T resistance could not be determined. Further studies are required to assess the expression and functionality of the detected genes in the studied isolate population to predict the phenotype consequences of the C/T resistant genotype.
Limitations of the study: (i) C/T was only evaluated for blood cultures, (ii) isolates and data originated from sentinel public hospitals in SA, (iii) small sample size, and (iv) not all the C/T resistant isolates could be sequenced due to limited funding.

4. Materials and Methods

4.1. Study Setting

A total of 100 E. coli, 100 K. pneumoniae, and 100 P. aeruginosa blood culture isolates were chronologically selected from storage (−70 °C). A three-week exclusion period was applied to avoid duplicate isolates of the same organism from the same patient.
The isolates were initially collected for the Group for Enteric, Respiratory, and Meningeal Diseases Surveillance (GERMS) program in SA (2010 to 2020), which receives clinical isolates from sentinel sites in the Free State, Gauteng, KwaZulu-Natal, Eastern- and Western Cape provinces. Demographic and clinical information of patients was collected by surveillance officers through medical record reviews and/or patient interviews using standard case report forms (CRFs). All GERMS-SA isolates were initially processed and stored as followed: the bacterial cultures were grown on blood or chocolate agar plates (Diagnostic Media Products (DMP), National Health Laboratory Service (NHLS), Johannesburg, SA) for no more than 18–27 h. The agar plates were carefully inspected for any contaminating bacterial or fungal colonies. Using a sterile disposable Pasteur pipette, 1 mL of TSB + 10% glycerol (DMP, NHLS, Johannesburg, SA) was dispensed into cryovials. Using a sterile swab or loop, a heavy sweep of growth was taken and emulsified in the cryovial containing the TSB + 10% glycerol. The cryovials were tightly sealed and placed in allocated cryoboxes and immediately stored at −70 °C.

4.2. Phenotypic Characterisation

All organisms were processed by the Centre for Healthcare-associated infections, Antimicrobial Resistance and Mycoses (CHARM), National Institute for Communicable Diseases (NICD), a division of the NHLS, Johannesburg, SA. The selected GERMS-SA isolates were retrieved from −70 °C storage. A loop full of the isolate + TSB + 10% glycerol mixture was streaked and grown on blood agar plates (DMP, NHLS, Johannesburg, SA) for no more than 18 h to 24 h. The agar plates were carefully inspected for any contaminating bacterial or fungal colonies. Organism identification was confirmed using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) (Microflex, Bruker Daltonics, Bremen, Germany). All MALDI-TOF MS score values were between 2.00 to 3.00 (high confidence identification) and fell in the consistency category A (high consistency). Antimicrobial susceptibility testing (AST) was performed using the Sensititre instrument (Trek Diagnostic Systems, East Grinstead, UK) with the commercially available Gram-negative DKMGN panel (Separation Scientific, Johannesburg, SA) which included amikacin, amoxicillin/clavulanic acid, aztreonam, cefotaxime, ceftazidime, C/A, C/T, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, tigecycline, tobramycin, and trimethoprim/sulfamethoxazole. The AST results were interpreted using the 2021 Clinical Laboratory Standards Institute (CLSI) guidelines (no interpretation for tigecycline was provided) [21]. MDR isolates were defined as non-susceptibility to one or more antibiotic agents in three or more antimicrobial classes [22]. XDR isolates were defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two categories), and PDR isolates were defined as non-susceptible to all agents in all antimicrobial categories [22].

4.3. Molecular Characterisation

Half (50%) of the C/T resistant isolates were randomly selected for WGS (i.e., 8 E. coli, 14 K. pneumoniae, and 7 P. aeruginosa isolates). The genomic DNA (gDNA) of the selected isolates was extracted with the QIAamp mini kit (Qiagen, Hilden, Germany) with the inclusion of lysozyme (10 mg/mL; Sigma-Aldrich, MS, USA) to ensure sufficient lysis. The quantity of the extracted gDNA was determined on Qubit 4.0 (Thermo Scientific, Waltham, MA, USA). Multiplexed paired-end libraries were prepared using the Nextera DNA Prep kit, followed by sequencing (2 × 150 bp) on a NextSeq 550 instrument (Illumina, Inc., San Diego, CA, USA) with 100× coverage at the NICD Sequencing Core Facility, Johannesburg, SA. Raw paired-end reads were analysed using the Jekesa pipeline (v1.0; https://github.com/stanikae/jekesa (accessed on 27 January 2022)).
Briefly, Trim Galore! (v0.6.2; https://github.com/FelixKrueger/TrimGalore (accessed on 27 January 2022)) was used to filter the paired-end reads (Q > 30 and length > 50 bp) [23]. De novo assembly was performed using SKESA v2.3.0 (https://github.com/ncbi/SKESA/releases (accessed on 27 January 2022)) and the assembled contigs were polished using Shovill (v1.1.0; https://github.com/tseemann/shovill (accessed on 27 January 2022)) [24,25]. Assembly metrics were calculated using QUAST (v5.0.2; http://quast.sourceforge.net/quast (accessed on 27 January 2022)) [26]. The assembled genome files were submitted to the National Center for Biotechnology Information GenBank and are available under BioProject number: PRJNA819852.
The multilocus sequence typing (MLST) profiles were determined using the MLST tool (version 2.16.4; https://github.com/tseemann/mlst (accessed on 20 September 2022)) [27]. Antimicrobial resistance (AMR) gene search was performed using ABRicate (version 1.0.1; https://github.com/tseemann/ABRicate (accessed on 20 September 2022)), against the Comprehensive Antibiotic Resistance Database (CARD), CARD-prevalence, Virulence Factor Database (VFDB) and ResFinder—Center for Genomic Epidemiology (CGE) database, with a gene alignment coverage cut-off of ≥95% and blastn sequence similarity of ≥95% [28,29,30,31,32,33].

4.4. Statistical Analysis

Microsoft Excel (version 2016) was used for data entry and basic statistical analysis (medians, interquartile ranges and percentiles). Additional data analyses were carried out using STATA statistical software package (version 14; StataCorp LP, USA).

5. Conclusions

Antibiotic resistance is a global health threat that limits the optimal care of patients infected with healthcare-associated pathogens. The increasing antibiotic resistance should be closely monitored. Since these isolates were not exposed to C/T previously, it is evident that E. coli, K. pneumoniae, and P. aeruginosa can adapt pre-existing resistance mechanisms, such as the production of catalytic enzymes (i.e., ESBLs and carbapenemases), altered PBPs, reduction in outer membrane channels resulting in decreased porin activity influx/expression, and an increase in efflux pumps to resist β-lactam molecules and inhibitors. In this study, C/T was not effective against the majority of MDR isolates.

Author Contributions

Conceptualisation, O.P. and A.S.-M.; methodology, M.L. and A.S.-M.; software, M.L.; validation, M.L. and A.S.-M.; formal analysis, M.L.; investigation, M.L.; resources, O.P.; data curation, M.L.; writing—original draft preparation, A.S.-M.; writing—review and editing, O.P., A.S.-M. and M.L.; visualisation, M.L.; supervision, M.L.; project administration, A.S.-M. and M.L.; funding acquisition, A.S.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Health Laboratory Service Research Trust (GRANT00494739).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the University of the Witwatersrand (Protocol No.: M190430 and M10464; date of approval: 2019).

Informed Consent Statement

Patient consent was waivered since patient care was not influenced at any stage.

Data Availability Statement

The datasets presented in this study can be found in the article or in the Appendix A.

Acknowledgments

We thank Nompumelelo Shezi, and Naseema Bulbulia for their assistance with the laboratory work. We would also like to thank the staff members of the Sequencing Core Facility (SCF) at the NICD for sequencing our isolate selection. We also thank the Institute Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at http://bigsdb.pasteur.fr/ (accessed on 23 March 2022).

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table
Table A1. Antibiotic resistance genes and plasmids detected in the sequenced Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa blood culture isolates.
Table A1. Antibiotic resistance genes and plasmids detected in the sequenced Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa blood culture isolates.
Isolate IDESCCO-ML0045ESCCO-016ESCCO-10882ESCCO-ML0129ESCCO-10151ESCCO-10377ESCCO-9385ESCCO-9889KLEPN-0028KLEPN-0203KLEPN-0098KLEPN-0024KLEPN-0196KLEPN-0004KLEPN-0051KLEPN-0062KLEPN-0063KLEPN-0109KLEPN-0192KLEPN-0036KLEPN-0175KLEPN-0097PSEAE-7954PSEAE-7985PSEAE-7981PSEAE-8174PSEAE-8183PSEAE-7986PSEAE-7904
Year20122020201620132015201520162016201020102010201020102010201020102010201020102010201020102014201420142014201420142014
ST10131405405410410410410137731011012313073073073073073363366065111111233233233273815
ProvinceGAGAGAGAGAGAGAGAWCKZWCGAGAWCGAGAGAGAGAWCGAGAGAWCGAGAWCGAGA
WardAAAAAAAAPPAAAAPAPAPPPPAAAPAPU
Antibiotic resistance genesaadA1 NNNNNNNNNYYYYNNYNNNNYNNNNNNNN
aadA2NNNNNNNNNNNNNNNNNNNNNYNNYYYNY
aadA5 NYYYYYYYNNNNNNNNNNNNNNNNNNNNN
aadA16NNNNNNNNNNNNNYYYNYYNNNNNNNNNN
aac (3)-IIa YNNNNNNNNYYYYYYYYYYYYYNNNNNNN
aac (3)-Id NNNNNNNNNNNNNNNNNNNNNNNNNYNNY
aac (3)-IId NNNYNNNNYNNNNNNNNNNYNNNNNNNNN
aac (6′)-Ib-cr YNYYYYYYNNYNYYYNYYYYYYNNNNNNN
aac (6′)-29a NNNNNNNNNNNNNNNNNNNNNNYYNNNNN
aac (6′)-Ib3 NNNNNNNNNNNNNNNNNNNNNNNNNNNYN
aac (6′)-Ig NNNNNNNNNNNNNNNNNNNNNNNNNNNYN
aac (6′)-Il NNNNNNNNNNNNNNNNNNNNNNNNYYYNY
aac (6′)-IIc NNNNNNNNNNNNNNNNNNNNNNNNNNNYN
aph (3′′)-IbYYYYYYYYNYYYYYYYYYYNYNNNNNNNN
aph (3′)-IIbNNNNNNNNNNNNNNNNNNNNNNYYYYYYY
aph (3′′)-Ib NNNNNNNNNNNNNNNNNNNNNNNNNNYYN
aph (6)-Id YYYYYYYYNYYYYYYYYYYYYNNNYNYYN
rmtBNNYNNNNNNNNNNNNNNNNNNNNNNNNNN
blaCMY-2YNNNYYYYNNNNNNNNNNNNNNNNNNNNN
blaCMY-4NNNNNNNNNNNNNNNNNNYNNNNNNNNNN
blaCTX-M-15YNYYYYYYYYYYYYYYYYYYYYNNNNNNN
blaCTX-M-27NYNNNNNNNNNNNNNNNNNNNNNNNNNNN
blaNDM-5NNYNNNNNNNNNNNNNNNNNNNNNNNNNN
blaNPSNNNNNNNNNNNNNNNNNNNNNNNNNNNYN
blaOXA-1YNYYYYYYNYYYYNYYYNYYYYNNNNNNN
blaOXA-4NNNNNNNNNNNNNNNNNNNNNNNNYYYNN
blaOXA-9NNNNNNNNNYNYYNNYNNNNNNNNNNNNN
blaOXA-181 NNNNYYYYNNNNNNNNNNNNNNNNNNNNN
blaOXA-395NNNNNNNNNNNNNNNNNNNNNNYYNNNNY
blaOXA-485NNNNNNNNNNNNNNNNNNNNNNNNNNNYN
blaOXA-486NNNNNNNNNNNNNNNNNNNNNNNNYYYNN
blaOXA-488NNNNNNNNNNNNNNNNNNNNNNNNNNNYN
blaPAONNNNNNNNNNNNNNNNNNNNNNYYYYYYY
blaSCO-1 NNNNNNNNNNYNNNNYNYNNYNNNNNNNN
blaSHV-1 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-26NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-28NNNNNNNNNNNYYYYYYYYNNNNNNNNNN
blaSHV-36NNNNNNNNNNNNNNNNNNNNNYNNNNNNN
blaSHV-40NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
blaSHV-56NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
blaSHV-78 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-79NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
blaSHV-85NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
blaSHV-89NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
blaSHV-94NNNNNNNNNNNNNNNNNNNYYNNNNNNNN
blaSHV-96NNNNNNNNNNNNNNNNNNNYYNNNNNNNN
blaSHV-98 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-106NNNNNNNNNNNYYYYYYYYNNNNNNNNNN
blaSHV-145 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-161 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-172NNNNNNNNNNNNNNNNNNNYYNNNNNNNN
blaSHV-179NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-187NNNNNNNNYNNNNNNNNNNNNNNNNNNNN
blaSHV-194 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaSHV-199 NNNNNNNNNNYNNNNNNNNNNNNNNNNNN
blaTEM-1ANNNNNNNNNNNNYNNNNNNNNNNNNNNNN
blaTEM-1B NNYYYYYYYYYNNYYYYYYYYYNNNNNNN
blaTEM-1CNNNNNNNNNNNNNNNNNNNNNYNNNNNNN
blaVIM-2NNNNNNNNNNNNNNNNNNNNNNYYYYYNY
fosANNNNNNNNYYYYYYYYYYYYYNYYYYYYY
ereANNNNNNNNNNYNNNNYNNNNNNNNNNNNN
mphA YYYYYYYYNNNNNNNNNNNNYYNNNNNNN
mdfA YYYYYYYYNNNNNNNNNNNNNNNNNNNNN
catA1NNNNNNNNNNNNNNNNNNNNYNNNNNNNN
catA2 NNNNNNNNYNNYYNYYNYNYNYNNNNNNN
catB3 YNYYYYYYNYYYYNYYYNYYYYNNNNNNN
catB7NNNNNNNNNNNNNNNNNNNNNNYYYYYYY
cmlA1 NNNNNNNNNYYNNNNYNNNNNNNNYYYNN
crpPNNNNNNNNNNNNNNNNNNNNNNYYYYYYY
floR NNNNNNNNNNNNNNNNNNYNNNNNNNNNN
OqxANNNNNNNNYYYYYYYYYYYYYNNNNNNNN
OqxBNNNNNNNNYYYYYYYYYYYYYNNNNNNNN
qnrB1NNNNNNNNNYNNNNNNNNNNNNNNNNNNN
qnrB6 NNNNNNNNNNNNNYYYNYNNNNNNNNNNN
qnrS1 YNNYYYYYNNNNNNNNNNYNNNNNNNNNN
ARR-2NNNNNNNNNYYNNNNNNNNNNNNNNNNNN
ARR-3 NNNNNNNNNNNNNYYNNYYNNNNNNNNNN
ARR-5NNNNNNNNNNNNNNNNNNNNNNNNYNYNN
sul1NYYYYYYYNYYNYYYYNYYNYYYYYYNYY
sul2 YYYYYYYYYYYYYYYYYYYYYYNNNNNNN
tetANYYNNNNNNYNNNNNNNNNYNNNNNNNNN
tetBNNNYYYYYNNNNNNNNNNNNNNNNNNNNN
tetDNNNNNNNNNNYYYNYYYYNNNNNNNNNNN
tetGNNNNNNNNNNNNNNNNNNNNNNNNYYYYN
dfrA1NNNNNNNNNNNNYNNNNNNNNNNNNNNNN
dfrA12NNNNNNNNNNNNNNNNNNNNNYNNNNNNN
dfrA14 YNNNYYYYNYYYYNNNYNYYYNNNNNNNN
dfrA15NNNNNNNNNNNNNNNNNNNNYNNNNNNNN
dfrA17 NYYYYYYYNNNNNNNNNNNNNNNNNNNNN
dfrA27NNNNNNNNNNNNNYYYNYYNNNNNNNNNN
dfrA30NNNNNNNNYNNNNNNNNNNYNNNNNNNNN
dfrB5NNNNNNNNNNNNNNNNNNNNNNNNYYYNY
OqxA NNNNNNNNYYYYYYYYYYYYYNNNNNNNN
OqxB NNNNNNNNYYYYYYYYYYYYYNNNNNNNN
qacENYYYYYYYNYYNYYYYNYYNYYYYYYYYY
PlasmidsCol(BS512)NNNNYYYYNNNNNNNNNNNNNNNNNNNNN
Col(MG828)YYYNNNNNNNNNNNNNYNYNNNNNNNNNN
Col156NYNNNNNNNNNNNNNNNNNNNNNNNNNNN
Col440INNNNNNNNNYYNNYNYNNNNNYNNNNNNN
Col440IINNNNNNNNNNNYYNNNNNNNNNNNNNNNN
ColKP3NNNNYYYYNNNNNNNNNNNNNNNNNNNNN
ColRNAIYYNNNNNNNYNNYNNNYNYYNNNNNNNNN
IncA/C2NNNNNNNNNNNNNNNNNNYNNNNNNNNNN
IncB/O/K/ZNNNNYYYYNNNNNNNNNNNNNNNNNNNNN
IncFIAYYYYYYYYNNNNNNNNNNNNNNNNNNNNN
IncFIA (HI1)NNNNNNNNYNYNNNYYNYNNNNNNNNNNN
IncFIB (AP001918)YYYYYYYYNNNNNNNNNNNNNNNNNNNNN
IncFIB (K)NNNNNNNNNYYYYYYYYYYYYNNNNNNNN
IncFIB (Mar)NNNNNNNNNNNNNNNNNNNNYYNNNNNNN
IncFIB (pQil)NNNNNNNNNNNNNNYNNNNNYNNNNNNNN
IncFIIYNYNNNNNNYNNNNNNNNNYNNNNNNNNN
IncFII (K)NNNNNNNNYYYYYYYYYYYYYNNNNNNNN
IncFII (pAMA1167-NDM-5)NNNYYYYYNNNNNNNNNNNNNNNNNNNNN
IncFII (pCoo)NNNNYYYYNNNNNNNNNNNNNNNNNNNNN
IncFII (pCRY)NNNNNNNNNNNYNNNNNNNNNNNNNNNNN
IncFII (pKPX1)NNNNNNNNNNNNNNNNNNNNNYNNNNNNN
IncFII (pRSB107)NYNNYYYYNNNNNNNNNNNNNNNNNNNNN
IncHI1BNNNNNNNNNNNNNNNNNNNNYNNNNNNNN
Incl1YNNNNNNNNNNNNNNNNNNNNNNNNNNNN
IncNNNNNNNNNNNNNNNNNNNYNNNNNNNNNN
IncQ1NNNYNNNNNNNNNNNNNNNYNNNNNNNNN
IncRNNNNNNNNYNYYYYYYNYNYYNNNNNNNN
IncX3NNNNYYYYNNNNNNNNNNNNNNNNNNNNN
IncX4YNNNYYYYNNNNNNNNNNNNNNNNNNNNN
p0111NNYNNNNNNNNNNNNNNNNNNNNNNNNNN
Green colour/N = No; Red colour/Y = Yes; GA = Gauteng; KZ = KwaZulu Natal; WC = Western Cape; A = Adult ward; P = Paediatric ward; U = Unknown ward.
Table
Table A2. Antibiotic susceptibility profiles of 8 Escherichia coli, 14 Klebsiella pneumoniae, and 7 Pseudomonas aeruginosa blood culture isolates selected for WGS.
Table A2. Antibiotic susceptibility profiles of 8 Escherichia coli, 14 Klebsiella pneumoniae, and 7 Pseudomonas aeruginosa blood culture isolates selected for WGS.
Amoxicillin/Clavulanate Acid
n = (%)		Ceftazidime/Avibactam
n = (%)		Ceftolozane/Tazobactam
n = (%)		Piperacillin/Tazobactam
n = (%)		Cefotaxime
n = (%)		Ceftazidime
n = (%) 		Aztreonam
n = (%) 		Ertapenem
n = (%)		Imipenem
n = (%) 		Meropenem
n = (%)		Colistin
n = (%) 		Gentamicin
n = (%)		Tobramycin
n = (%)		Amikacin
n = (%) 		Ciprofloxacin
n = (%)		Trimethoprim/Sulfamethoxazole
n = (%)
E. coli (n = 8)S1
(13)		6
(75)		0
(0)		1
(13)		0
(0)		0
(0)		0
(0)		0
(0)		3
(38)		3
(38)		0
(0)		4
(50)		0
(0)		3 (38)0
(0)		0
(0)
I0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		1
(13)		0
(0)		7
(77)		0
(0)		0
(0)		2
(25)		0
(0)		0
(0)
R7
(88)		2
(25)		8
(100)		7
(88)		8
(100)		8
(100)		8
(100)		8
(100)		4
(50)		5
(63)		1
(13)		4
(50)		8
(100)		3
(38)		8
(100)		8
(100)
K. pneumoniae (n = 14)S0
(0)		14 (100)0
(0)		1
(7)		0
(0)		0
(0)		0
(0)		14 (100)14 (100)14 (100)0
(0)		0
(0)		1
(7)		14 (100)2
(14)		0
(0)
I0
(0)		0
(0)		0
(0)		1
(7)		0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		0
(0)		13 (93)0
(0)		2
(14)		0
(0)		1
(7)		0
(0)
R14 (100)0
(0)		14 (100)12 (86)14 (100)14 (100)14 (100)0
(0)		0
(0)		0
(0)		1
(7)		14 (100)11
(79)		0
(0)		11
(79)		14
(100)
P. aeruginosa (n = 7)S0 *
(0)		0
(0)		0
(0)		0
(0)		0 *
(0)		0
(0)		6
(84)		0 *
(0)		0
(0)		0
(0)		0
(0)		1
(14)		0
(0)		0
(0)		0
(0)		0 *
(0)
I0 *
(0)		0
(0)		0
(0)		1
(14)		0 *
(0)		0
(0)		1
(14)		0 *
(0)		0
(0)		0
(0)		4
(57)		1
(14)		0
(0)		0
(0)		0
(0)		0 *
(0)
R0 *
(0)		7
(100)		7
(100)		6
(86)		0 *
(0)		7
(100)		0
(0)		0 *
(0)		7
(100)		7
(100)		3
(43)		5
(71)		7
(100)		7
(100)		7
(100)		0 *
(0)
S = Susceptible; I = Intermediate; R = Resistant; * = Antibiotic MIC breakpoints not covered in the 2021 Clinical Laboratory Standards Institute (CLSI) guidelines.

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Table
Table 1. Demographic characteristics of patients with Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa bacteraemia.
Table 1. Demographic characteristics of patients with Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa bacteraemia.
Demographic CharacteristicsE. coliK. pneumoniaeP. aeruginosa
n = 100n = 100n = 100
Age
Median age (interquartile range (IQR))36 years
(21–54 years)		31 years
(130 days–51 years)		44 years
(11–58 years)
Sex
Female613641
Male376055
Unknown244
Ward
Adult745769
Paediatric 244025
Unknown236
Province
Eastern Cape1--
Free State-102
Gauteng986546
Western Cape-2441
KwaZulu-Natal1110
Unknown--1
Table
Table 2. Antibiotic susceptibility profiles of 100 Escherichia coli, 100 Klebsiella pneumoniae and 100 Pseudomonas aeruginosa blood culture isolates.
Table 2. Antibiotic susceptibility profiles of 100 Escherichia coli, 100 Klebsiella pneumoniae and 100 Pseudomonas aeruginosa blood culture isolates.
Amoxicillin/Clavulanate acidCeftazidime/AvibactamCeftolozane/TazobactamPiperacillin/TazobactamCefotaximeCeftazidimeAztreonamErtapenemImipenemMeropenemColistinGentamicinTobramycinAmikacinCiprofloxacinTrimethoprim/SulfamethoxazoleMDR Detected
E. coliS369682776270617989910887489442530
I180210542319402780
R464162238253519886122444875
K. pneumoniaeS33100665135363697991000413999433461
I1061302021094010150
R66028366462641006595105266
P. aeruginosaS0 *8485670 *75760 *65710787886730 *24
I0 *02100 *6110 *896832210 *
R0 *1613230 *19130 *272032192012260 *
S = Susceptible; I = Intermediate; R = Resistant; MDR = Multidrug resistance; * = Antibiotic MIC breakpoints not covered in the 2021 Clinical Laboratory Standards Institute (CLSI) guidelines.
Table
Table 3. Antibiotic resistance genes detected in the sequenced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa blood culture isolates.
Table 3. Antibiotic resistance genes detected in the sequenced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa blood culture isolates.
Antibiotic ClassOrganismAntibiotic Resistance Genen =ST
AminoglycosideE. coliaadA57131; 405; 410
aac (3)-IIa110
aac (3)-Iid1405
aac (6′)-Ib-cr710; 405; 410
aph (3′′)-Ib; aph (6)-Id810; 131; 405; 410
rmtB1405
K. pneumoniaeaac (3)-Iia1337; 73; 101; 231; 307; 336; 6065 *
aac (6′)-Ib-cr1073; 101; 231; 307; 336; 6065 *
aadA1637; 73; 101; 307; 336
aadA165231; 307
aadA216065 *
aph (3′′)-Ib1137; 73; 101; 231; 307; 336
aph (6)-Id1237; 73; 101; 231; 307; 336
aac (3)-Iid21; 336
P. aeruginosaaac (3)-Id2233; 815
aac (6′)-29a2111
aac (6′)-Ib3; aac (6′)-Ig; aac (6′)-Iic1273
aac (6′)-Il; aadA24233; 815
aph (3′’)-Ib2233; 273
aph (3′)-Iib7111; 233; 273; 815
aph (6)-Id3233; 273
β-lactamE. coliblaCMY-2510; 410
blaCTX-M-15710; 405; 410
blaCTX-M-271131
blaNDM-51405
blaOXA-1710; 405; 410
blaOXA-1814410
blaTEM-1B6405; 410
K. pneumoniaeblaCTX-M-151437; 73; 101; 231; 307; 336; 6065 *
blaCMY-41307
blaOXA-11137; 73; 101; 307; 336; 6065 *
blaOXA-9437; 101; 307
blaSCO-1473; 307; 336
blaSHV-1; blaSHV-26173
blaSHV-288101; 231; 307
blaSHV-3616065 *
blaSHV-40; blaSHV-56; blaSHV-79; blaSHV-85; blaSHV-89137
blaSHV-94; blaSHV-96; blaSHV-1722336
blaSHV-78; blaSHV-98;blaSHV-145; blaSHV-161; blaSHV-179; blaSHV-194; blaSHV-199173
blaSHV-1068101; 231; 307
blaSHV-18711
blaTEM-1A1101
blaTEM-1B121; 37; 73; 231; 307; 336; 6065 *
blaTEM-1C16065
P. aeruginosablaNPS1273
blaOXA-43273
blaOXA3953111; 815
blaOXA-485; blaOXA-4881273
blaOXA-4863273
blaPAO7111; 233; 273; 815
blaVIM-26111; 233; 815
FosfomycinE. coliNone detected--
K. pneumoniaefosA131; 37; 73; 101; 231; 307; 336
P. aeruginosafosA7111; 233; 273; 815
MacrolideE. colimdfA; mphA810; 131; 405; 410
K. pneumoniaeereA273; 307
mphA2336; 6065 *
P. aeruginosaNone detected--
PhenicolE. colicatB3710; 405; 410
K. pneumoniaecatA11336
catA281; 101; 307; 336; 6065 *
catB31137; 101; 307; 336; 6065 *
cmlA1337; 73; 307
floR1307
P. aeruginosacatB77111; 233; 273; 815
cmlA13233
QuinoloneE. coliaac (6′)-Ib-cr710; 405; 410
qnrS1610; 405; 410
K. pneumoniaeOqxA; OqxB131; 37; 73; 101; 231; 0307; 336
aac (6′)-Ib-cr1173; 101; 231; 307; 336; 6065 *
qnrB1; qnrS1137
qnrB64231; 307
P. aeruginosaaac (6′)-Ib-cr1273
crpP7111; 233; 273; 815
RifampicinE. coliNone detected--
K. pneumoniaeARR-2237; 73
ARR-34231; 307
P. aeruginosaARR-52233
SulphonamideE. colisul17131; 405; 410
sul2810; 131; 405; 410
K. pneumoniaesul11037; 73; 101; 231; 307; 336; 6065 *
sul2141; 37; 73; 101; 231; 307; 336; 6065 *
P. aeruginosasul16111; 233; 273; 815
TetracyclineE. colitetA2131; 405
tetB5405; 410
K. pneumoniaetetA237; 336
tetD773; 101; 307
P. aeruginosatetG4233; 273
TrimethoprimE. colidfrA14510; 410
dfrA177131; 405; 410
K. pneumoniaedfrA11101
dfrA1216065 *
dfrA14837; 73; 101; 307; 336
dfrA151336
dfrA275231; 307
dfrA3021; 336
P. aeruginosadfrB54233; 815
DisinfectantE. colisitABCD410; 131; 405
qacE7131; 405; 410
K. pneumoniaeOqxA; OqxB131; 37; 73; 101; 231, 307; 336
qacE1037; 73; 101; 231; 307; 336; 6065 *
P. aeruginosaqacE7111; 233; 273; 815
ST = Sequence type; * = Novel ST detected.
Table
Table 4. Plasmids detected in the sequenced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa blood culture isolates.
Table 4. Plasmids detected in the sequenced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa blood culture isolates.
OrganismSTPlasmidTotal Number of Isolates
E. coli10IncFIB (AP001918); ColRNAI; Col (MG828); IncFIA; IncX4; IncFII; IncI11
131IncFII (pRSB107); IncFIB (AP001918); ColRNAI; Col156; Col (MG828); IncFIA;1
405IncFII (pAMA1167-NDM-5) *; IncFIB (AP001918); Col (MG828) *; IncFIA; p0111 *; IncFII *; IncQ1 *2
410IncFII (pRSB107); IncB/O/K/Z; IncFII (pAMA1167-NDM-5); IncFIB (AP001918); IncFIA; Col (BS512); ColKP3; IncX4; IncX3; IncFII (pCoo)4
K. pneumoniae1IncFII (K); IncR; IncFIA (HI1)1
37IncFII (K); Col440I; IncFIB (K); ColRNAI; IncFII1
73IncFII (K); Col440I; IncR; IncFIB (K); IncFIA (HI1)1
101IncFII (K); IncR; IncFIB (K); Col440II; IncFII (pCRY) *; ColRNAI *2
231IncFII (K); Col440I; IncR; IncFIB (K)1
307IncFII (K); Col440I *; IncR *; IncFib (K); IncFIB (pQil) *; IncFIA (HI1) *;
ColRNAI *; Col (MG828) *; IncN *; IncA-C2 *		5
336IncFII (K); IncR; IncFIB (K); IncFIB (pQil) *; ColRNAI *; IncFIB (Mar) *; IncFII *; IncHI1B *; IncQ1 *2
6065Col440I; IncFib (Mar); IncFII (pKPX1)1
P. aeruginosaST111-0
ST233-0
ST273-0
ST815-0
* = Plasmids not detected in all isolates; - = No plasmid/s detected.
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Perovic, O.; Singh-Moodley, A.; Lowe, M. In Vitro Activity of Ceftolozane-Tazobactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Obtained from Blood Cultures from Sentinel Public Hospitals in South Africa. Antibiotics 2023, 12, 453. https://doi.org/10.3390/antibiotics12030453

AMA Style

Perovic O, Singh-Moodley A, Lowe M. In Vitro Activity of Ceftolozane-Tazobactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Obtained from Blood Cultures from Sentinel Public Hospitals in South Africa. Antibiotics. 2023; 12(3):453. https://doi.org/10.3390/antibiotics12030453

Chicago/Turabian Style

Perovic, Olga, Ashika Singh-Moodley, and Michelle Lowe. 2023. "In Vitro Activity of Ceftolozane-Tazobactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Obtained from Blood Cultures from Sentinel Public Hospitals in South Africa" Antibiotics 12, no. 3: 453. https://doi.org/10.3390/antibiotics12030453

APA Style

Perovic, O., Singh-Moodley, A., & Lowe, M. (2023). In Vitro Activity of Ceftolozane-Tazobactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa Obtained from Blood Cultures from Sentinel Public Hospitals in South Africa. Antibiotics, 12(3), 453. https://doi.org/10.3390/antibiotics12030453

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