The emergence of antibiotic resistance is increasing and represents one of the major threats to global health today. Infections caused by multidrug resistant microorganisms cause relevant clinical and economic impact leading to increased hospital stays, costs and mortality.1 The Centers for Disease Control and Prevention (CDC) points that about two million people in the USA present with infections caused by bacteria resistant to at least one antimicrobial agent of choice each year, leading to at least 23,000 deaths annually.2 Healthcare-associated infections account for a high incidence of multidrug resistant microorganisms due to recurrent overuse and misuse of these medications, high density of susceptible hosts, and increased odds of transmission among patients.3,4

In 2017, the World Health Organization (WHO) published a list of pathogens with priority demand for new antibiotics due to antimicrobial resistant profile. Amongst the critical priority pathogens were carbapenem-resistant Pseudomonas aeruginosa, and carbapenem and third-generation cephalosporins-resistant Enterobacterales, which includes Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Serratia spp., Proteus spp., Providencia spp., and Morganella spp.5

P. aeruginosa has a leading role among infections caused by Gram-negative rods, especially in critically ill and immunocompromised patients, e.g. those who experienced extensive, high-degree burns.6 Treatment options are limited due to antimicrobial resistance, which permitted P. aeruginosa infections to become a serious issue causing a total of 51,000 healthcare-associated infections per year in the USA.7,8 In Brazil, P. aeruginosa is the fifth bacterial pathogen in bloodstream infections associated with central venous catheters in adult patients, accounting for 8.5% of this infection in 2016.9

Currently, eight categories of antibiotics are mainly used to treat P. aeruginosa infections such as aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillin with β-lactamase inhibitors, monobactams, fosfomycin, and polymyxin. However, Gram-negative bacilli like P. aeruginosa may present many mechanisms of antibiotic resistance, which can be expressed at the same time, including decrease in influx porin activity, increase in efflux pumps, production of altered penicillin-binding proteins and catalytic enzymes that inactivate antibiotics.7,10

Meanwhile, Enterobacterales also represent a significant concern due to carbapenem-resistance.11 Currently, therapies against carbapenem-resistant Enterobacterales include tigecycline, ceftazidime-avibactam, and other drugs not widely used recently such as colistin, fosfomycin, and aminoglycosides.11

Few studies have reported the profile of new antibiotics in low-to middle income countries. Pfaller et al. reported the in vitro activity of ceftolozane–tazobactam and comparator agents against Latin American isolates Enterobacterales and P. aeruginosa from 2013 to 2015.12 Considering the need for updated knowledge about susceptibility profile, especially regarding new antibiotics, this study aimed to evaluate the in vitro activity of ceftolozane–tazobactam against clinical bacterial isolates of P. aeruginosa, E. coli, K. pneumoniae, and other Enterobacterales from Brazil.

A total of 673 isolates from 10 sites were included during the study period. The clinical isolates were retrieved from intensive care units (ICU) (75%) and from non-ICU (25%).

Table 1 shows the antibiotics assessed, the range and concentration of each drug, and Table 2 shows the distribution of species tested. E. coli (N=216; 32.1%), K. pneumoniae (N=193; 28.7%), and P. aeruginosa (N=132; 19.6%) were the most prevalent species.

Table 3 shows the susceptibility profile of different organisms to several antibiotics.

Considering the analysis performed for P. aeruginosa, the susceptibility rate to ceftolozane–tazobactam was 84.9%, with MIC50/90 values of 1/16μg/mL (Table 3; Fig. 1). Colistin was the most active compound, with 99.2% of isolates susceptible (MIC50/90, ≤1/≤1μg/mL); cefepime, ciprofloxacin, and levofloxacin have also shown estimates above 60%.

Fig. 1.

Frequency distribution (n) of ceftolozane–tazobactam at each MIC (μg/mL) for 132 Pseudomonas aeruginosa from Brazil.

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Ceftolozane–tazobactam susceptibility rate against E. coli was 94.9%. Susceptibility rates to other antibiotics were 97.7% to amikacin, 97.2% to meropenem, 94.0% to ertapenem, and 91.7% to piperacillin-tazobactam. The activity of ceftolozane–tazobactam against E. coli was high (MIC50/90, 0.25/1μg/mL) (Table 3; Fig. 2).

Fig. 2.

Frequency distribution (n) of ceftolozane–tazobactam at each MIC (μg/mL) for 216 Escherichia coli from Brazil.

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Susceptibility rate of K. pneumoniae to ceftolozane–tazobactam was 40.4%, with weak activity against this microorganism (MIC50/90, 16/>32μg/mL), but higher susceptibility to amikacin (91.2%), colistin (90.2%), imipenem (61.1%), meropenem (60.1%), and ertapenem (53.4%) (Table 3; Fig. 3). Other antibiotics have shown inhibition frequencies that ranged from 20.7% to 39.9% (Table 3; Fig. 3).

Fig. 3.

Frequency distribution (n) of ceftolozane–tazobactam at each MIC (μg/mL) for 193 Klebsiella pneumoniae from Brazil.

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The analysis performed with other Enterobacterales have shown a susceptibility rate to ceftolozane–tazobactam of 81.1% (MIC50/90, 0.5/16μg/mL). These microorganisms had greater susceptibility to meropenem (93.9%), amikacin (90.9%), and ertapenem (88.6%) – Table 3; Fig. 4.

Fig. 4.

Frequency distribution (n) of ceftolozane–tazobactam at each MIC (μg/mL) for 132 other* Enterobacterales from Brazil.

* Other Enterobacterales consist of (n): Klebsiella aerogenes (3); E. asburiae (1); E. cloacae (38); Klebsiella oxytoca (11); K. variicola (4); Morganella morganii (1); Proteus mirabilis (72); P. vulgaris (1); Providencia rettgeri (1).

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From 13 ceftolozane/tazobactam non-susceptible P. aeruginosa isolates, carbapenemase was detected in 8/13 (62%) isolates which fully explains the detected resistance. The presence of at least one carbapenemase gene was demonstrated in only one isolate (KPC), while the identity of the carbapenemase gene harbored by the remaining 7/8 strains (87.5%) remains unknown and is yet to be studied.

From the remaining five (38%) isolates tested negative for the presence of carbapenemases by Carba-NP, three were randomly chosen to be sequenced aiming to detect previously reported mutations associated to the ceftolozane/tazobactam resistant phenotype.

As expected, genes encoding aminoglycoside modifying enzymes (aph(3′β-IIb)), chloramphenicol acetyltransferases (catB7), and fosfomycin resistance enzymes (fosA) were found in all three isolates. Regarding β-lactamases genes, blaPAO (blaAmpC) and blaOXA-50 were also detected in all isolates tested.

AmpC substitutions G27D, V205L, and G391A were all found in only one isolate, while substitution T105A was found in two isolates. On the other hand, AmpC substitutions E114A, G283E, and M288R were all detected in only one is olate.

This study was conducted aiming to evaluate the activity of ceftolozane–tazobactam against clinical bacterial isolates from Brazil. Thus, in vitro analyses were performed and susceptibility of different organisms to several antibiotics are presented.

Microorganisms tested have shown susceptibility rates to ceftolozane–tazobactam ranging from 40.4% (K. pneumoniae) to 94.9% (E. coli). The susceptibility profile to ceftolozane–tazobactam have varied according with microorganism by intrinsic and extrinsic mechanisms and also according to the country that provided the samples.12,16–27 Shortridge et al. assessed the activity of ceftolozane–tazobactam against Gram-negative isolates from bloodstream infection in the US from 2013 to 2015. In that study, E. coli NDM-1, K. pneumoniae NDM-1, and P. aeruginosa pan-β-lactams non-susceptible (pan-BL-NS) presented complete resistance. P. aeruginosa pan-BL-NS were non-susceptible to all β-lactams including ceftolozane–tazobactam. Isolates containing carbapenemases or metallo-beta-lactamases were uncommon (1.4%) in the study, which compromised the robustness of the findings.25 However, three studies have reported susceptibility to ceftolozane–tazobactam that reached 100%, against the following microorganisms: E. coli CTX-M 14-like, E. coli SHV ESBL, E. coli TEM ESBL, K. pneumoniae CTX-M 14-like, Citrobacter koseri, Proteus mirabilis and Proteus spp.23,25,28 Garcia-Fernandez et al. highlight the activity of ceftolozane–tazobactam against wild type Enterobacterales, although it is dependent on both the species and resistance mechanisms.28 In our study, the susceptibility of Klebsiella spp. to ceftolozane–tazobactam was low. This was due to high incidence of carbapenemases in this microorganism, most of them with high rate of hydrolysis of ceftolozane, like KPC. This result suggests that ceftolozane–tazobactam should be used cautiously in empirical therapies considering the high incidence of KPC-producing bacteria. It is important to note that MIC cut-off for Enterobacterales was 1mg/L, compared to 4mg/L for P. aeruginosa. This is due to higher affinity of ceftolozane to P. aeruginosa binding sites.

Regarding the observed susceptibility of P. aeruginosa, 84.9% were susceptible to ceftolozane–tazobactam. The same pattern was observed for other Enterobacterales, with susceptibility rates above 80%. These microorganisms represent a great concern for clinical management on community and hospital acquired infections due to its resistance to available antibiotics. Carbapenem resistance observed in Enterobacterales, usually mediated by transferable β-lactamase enzymes, has also become an important issue.29

Some authors have studied the activity of ceftolozane–tazobactam specifically against P. aeruginosa and Enterobacterales isolates from Germany, USA, Spain, and Latin America. The frequency of susceptibility rates ranged from 30% to 100% for P. aeruginosa and from 77.8% to 100% for Enterobacterales.12,18,20,23–26,28 Pfaller et al. reported results of ceftolozane–tazobactam activity against multidrug-resistant Enterobacterales and P. aeruginosa from Latin America, including isolates from Brazil. Ceftolozane–tazobactam was the most active of the β-lactam agents tested against P. aeruginosa and it was the second most active drug, after meropenem, against Enterobacterales.12

Garcia-Fernandez et al. reported the activity of ceftolozane–tazobactam specifically in the context of intensive care units, where a worse pattern of resistance is observed. When P. aeruginosa was assessed, a susceptibility rate of 91.3% was observed and it was the third most active, after colistin (95.0%) and amikacin (93.8%). When Enterobacterales were assessed, ceftolozane–tazobactam activity ranged from 5% (carbapenemase-phenotype K. pneumoniae) to 100% (Proteus spp.),28 which is aligned with the most recent national surveillance findings, where almost 45% of K. pneumoniae bloodstream infection isolates were resistant to 3rd and 4th generation cephalosporins and carbapenems.30

Some resistant isolates produce enzymes able to degrade or inactivate β-lactams, such as ESBL samples.31 Being a combination of a β-lactam and an inhibitor of a considerable number of β-lactamase enzymes, ceftolozane–tazobactam retains activity against isolates resistant to other β-lactams, sparing the use of meropenem.32–34 It is well known that carbapenem use results in escalating rates of carbapenem-resistant infections, limiting treatment options and increasing mortality.35

In this context, a sub-analysis was performed in order to describe genetic profile of resistant mechanisms in ceftolozane/tazobactam non-susceptible P. aeruginosa isolates. The presence of a carbapenemase in most of the isolates is consistent with results previously described in the country.36–39 Although one isolate had at least one carbapenemase gene, the remaining still needs further analysis to better address this issue. In Brazil, it is estimated that the frequency of carbapenem-resistant P. aeruginosa may reach 40.0% of the observed infections.39–42 The results shown in the present study highlights the presence of intrinsic mechanisms, and that not all cases of resistance were mediated by transferable carbapenemase-encoding genes. Ceftolozane–tazobactam resistance in non-carbapenemase producing isolates were associated with ampC over-expression by single point mutations in the blaPDC gene (ampC gene) identified by whole gene sequencing. AmpC substitutions G27D, V205L, G391A, T105A, E114A, G283E, and M288R were all associated with AmpC overexpression.

When compared to polymyxins and aminoglycosides, ceftolozane–tazobactam has a greater safety profile.1 Polymyxins and aminoglycosides are antibiotics with high frequency of toxicity and the last choice when modern antimicrobials are not effective or contraindicated1,43 In a meta-analysis, colistin showed a similar safety profile when compared with other antibiotics in patients with ventilator-associated pneumonia.44 However, numerous adverse events, such as mortality, nephrotoxicity, neurotoxicity, allergic and topical reactions, were related with the use of polymyxins, highlighting its toxicity.43 Thus, since ceftolozane–tazobactam has a favorable safety profile, it seems to be a good option in clinical practice.

One study limitation needs to be highlighted: the lack of an alternative test to confirm MIC results of the isolates susceptibility profile. Despite MIC testing recommendation for antimicrobial susceptibility assessment, the study would benefit from using an additional technique or different approach to further investigate that point. Even though, the study provides new information about the activity of this antimicrobial agent against different isolates from Brazil.

As a regional surveillance network that collected 2410 pathogens from selected medical centers, the study does not provide a population-based data, nor information about the incidence of infections in a given region.

In conclusion, ceftolozane–tazobactam has shown satisfactory activity against a great variety of microorganisms analyzed, especially when respiratory tract is the infection site as reported in clinical studies, showing promising results even against resistant strains.17,21,45,46

The activity of ceftolozane–tazobactam is specially observed against P. aeruginosa, E. coli, and other Enterobacterales. Our data suggest that ceftolozane–tazobactam may be an important treatment option, including against multidrug resistant strains.

Medical writing was provided by Roberta Arinelli Fernandes, Ana Carolina Padula Ribeiro Pereira and Ayke Adnet de Lima of Sense Company Brazil. This assistance was funded by MSD Brazil.

All authors were involved in at least one of the following: conception, design of work or acquisition, analysis, interpretation of data and drafting the manuscript and/or revising/reviewing the manuscript for important intellectual content. All authors provided final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Suellen da Silva Rodrigues, Fernando Brandão Serra, and Marina Della-Negra de Paula, are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ USA, who may own stock and/or hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA.

FFT received research grants from MSD, Pfizer and TEVA.

This study was supported by Merck Sharpe Dohme.