ABSTRACT
Both typhoidal and nontyphoidal salmonellae are included in the top 15 drug-resistant threats described by the U.S. Centers for Disease Control and Prevention. There is an urgent need to look for alternative antibiotics for the treatment of Salmonella infections. We used the broth microdilution test to examine the in vitro susceptibilities of typhoidal and nontyphoidal salmonellae, including isolates positive for extended-spectrum β-lactamase (ESBL), to ceftolozane/tazobactam and six other antibiotics. Of the 313 (52 typhoidal and 261 nontyphoidal) Salmonella isolates tested, 98.7% were susceptible to ceftolozane/tazobactam. Based on the overall MIC50/90 values, Salmonella isolates were more susceptible to ceftolozane/tazobactam (0.25/0.5 mg/L) than all the comparator agents: ampicillin (≥64/≥64 mg/L), levofloxacin (0.25/1 mg/L), azithromycin (4/16 mg/L), ceftriaxone (≤0.25/4 mg/L), chloramphenicol (8/≥64 mg/L), and trimethoprim/sulfamethoxazole (1/≥8 mg/L). Comparison of the activities of the antimicrobial agents against nontyphoidal Salmonella isolates according to their serogroups showed that ceftolozane/tazobactam had the highest activity (100%) against Salmonella serogroup D, G, I, and Q isolates, whereas the lowest activity (85.7%) was observed against serogroup E isolates. All 10 ESBL-producing Salmonella isolates (all nontyphoidal), of which 8 were CTX-M-55 producers and 2 were CTX-M-65 producers, were sensitive to ceftolozane/tazobactam, albeit with MIC50/90 values higher (1/2 mg/L) than those for non-ESBL producers (0.25/0.5 mg/L). In summary, our data indicate that ceftolozane/tazobactam is active against most strains of both typhoidal and nontyphoidal salmonellae and also against ESBL-producing salmonellae.
KEYWORDS: ceftolozane/tazobactam, Salmonella, typhoidal, nontyphoidal, ESBL, antimicrobial resistance
INTRODUCTION
Salmonellosis is associated with significant mortality and morbidity worldwide. It is estimated that nontyphoidal salmonellosis causes 94 million gastroenteritis cases and 0.16 million deaths globally per year, while typhoidal salmonellosis causes 21.7 million cases and 0.22 million deaths (1, 2). Traditionally, salmonella infections were treated with the conventional first-line drugs ampicillin, cotrimoxazole, and chloramphenicol. Since the 1990s, fluoroquinolones have been used widely for the treatment of both typhoidal and nontyphoidal salmonellosis. Due to the increasing incidence of antibiotic resistance, third-generation cephalosporins and azithromycin have now become the mainstay treatments for salmonella infections, although reports of resistance to these antibiotics are also on the rise. In particular, the emergence of antimicrobial resistance in salmonellae in India, Pakistan, and Bangladesh is of major concern (3–6). Recognizing the severity of this problem, the U.S. Centers for Disease Control and Prevention (CDC) included both typhoidal salmonellae and nontyphoidal salmonellae in its 2019 report on the top 15 drug-resistant threats (https://www.cdc.gov/drugresistance/biggest-threats.html). There is an urgent need to look for alternative antibiotics, particularly new antibiotics on the horizon, for the treatment of salmonella infections.
Ceftolozane/tazobactam is a new broad-spectrum cephalosporin/β-lactamase inhibitor combination recently launched in the market. Based on the results from its in vitro susceptibility spectrum and preliminary clinical experience, it is particularly useful for the treatment of infections associated with extended-spectrum β-lactamase (ESBL)-producing Enterobacterales, including Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (7–12). Recently, we have shown that ceftolozane/tazobactam is highly active against Burkholderia pseudomallei, a bacterium that is locally important in causing fatal systemic infections in Southeast Asia (13). Moreover, our preliminary data using Etest have shown that ceftolozane/tazobactam could be useful against Salmonella enterica serovar Typhi strains (14). Based on these data, we hypothesized that ceftolozane/tazobactam may be active against a significant proportion of typhoidal and nontyphoidal Salmonella strains. Since ceftolozane/tazobactam is active against E. coli and K. pneumoniae, which produce ESBL, we hypothesized that ESBL-producing salmonellae may also be susceptible to ceftolozane/tazobactam. To test this hypothesis, we examined the in vitro susceptibilities of 313 typhoidal and nontyphoidal Salmonella strains, including isolates that are ESBL positive, to ceftolozane/tazobactam and six other antibiotics.
RESULTS
Collection of Salmonella clinical isolates.
A total of 52 typhoidal and 261 nontyphoidal Salmonella isolates were collected from three hospitals in Hong Kong from January 2011 to July 2020. Of the 313 isolates, 285 were obtained from routine clinical stool samples and 28 were obtained from blood culture. The typhoidal isolates included Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B. The nontyphoidal isolates included S. Enteritidis, S. Typhimurium, S. Anatum, S. Bareilly, S. Derby, S. London, S. Poona, S. Rissen, S. Saintpaul, and S. Stanley, which were serogrouped as Salmonella groups B, C, D, E, G, I, and Q.
Detection of ESBL-producing Salmonella isolates.
Of the 313 Salmonella isolates, 10 (3.2%) were phenotypically confirmed to be ESBL producers. Their inhibition zones for cefotaxime (CTX)-clavulanic acid (CLA) and/or ceftazidime (CAZ)-CLA were enhanced by 5 to 8 mm over those for the respective antimicrobial disk alone. These 10 isolates were all nontyphoidal Salmonella strains, with 7 from serogroup B, 1 from serogroup C, 1 from serogroup D, and 1 from serogroup E. All the ESBL producers were obtained from blood culture.
Determination of ESBL-encoding genes.
The 10 ESBL-producing Salmonella isolates were all confirmed by PCR amplification to carry blaCTX-M. Following sequencing and comparison to known β-lactamase-encoding genes, eight isolates (six from serogroup B, one from serogroup D, and one from serogroup E) were determined to be CTX-M-55 producers, while two (one from serogroup B and one from serogroup C) were CTX-M-65 producers.
Antimicrobial susceptibilities of Salmonella clinical isolates.
The in vitro activities of ceftolozane/tazobactam and the six antimicrobial comparators against the Salmonella isolates are summarized in Tables 1 to 3. Based on the overall MIC50/90 values (Table 1), Salmonella isolates were more susceptible to ceftolozane/tazobactam (0.25/0.5 mg/L) than to all six comparator agents: ampicillin (≥64/≥64 mg/L), levofloxacin (0.25/1 mg/L), azithromycin (4/16 mg/L), ceftriaxone (≤0.25/4 mg/L), chloramphenicol (8/≥64 mg/L), and trimethoprim/sulfamethoxazole (1/≥8 mg/L). Of the 313 isolates tested, 98.7% (309/313) were susceptible to ceftolozane/tazobactam, representing 100% (52/52) and 98.5% (257/261) of the typhoidal and nontyphoidal isolates, respectively (Table 1). Susceptibility rates of >85% were observed for azithromycin (92.3%) and ceftriaxone (88.8%); however, their activities against typhoidal isolates (100.0% and 98.1%, respectively) were higher than those against nontyphoidal isolates (90.8% and 87.0%, respectively). In addition, the activity of levofloxacin was particularly more effective against typhoidal isolates (65.4% susceptible) than against nontyphoidal isolates (37.5% susceptible).
TABLE 1.
Cumulative MIC distributions, MIC50 and MIC90 values, and susceptibilities of Salmonella clinical isolates from Hong Kong to ceftolozane/tazobactam and six comparator agents
| Isolates (no.) and antimicrobial agenta | No. (cumulative %) of isolates inhibited at a MIC (mg/L) ofb: |
MIC (mg/L) |
Susceptibilityf (%) |
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.03c | 0.06c | 0.12c | 0.25c | 0.5c | 1c | 2c | 4 | 8d | 16d | 32d | 64d | Range | 50%e | 90%e | S | I | R | |
| All (313) | ||||||||||||||||||
| Ceftolozane/tazobactam | 91 (29.1) | 76 (53.4) | 122 (92.3) | 15 (97.1) | 5 (98.7) | 2 (99.4) | 1 (99.7) | 1 (100.0) | ≤0.12 to >16 | 0.25 | 0.5 | 98.7 | 0.6 | 0.6 | ||||
| Ampicillin | 22 (7.0) | 10 (10.2) | 13 (14.4) | 10 (17.6) | 8 (20.1) | 250 (100.0) | ≤2 to >64 | >64 | >64 | 14.4 | 3.2 | 82.4 | ||||||
| Levofloxacin | 91 (29.1) | 9 (31.9) | 32 (42.2) | 99 (73.8) | 49 (89.5) | 21 (96.2) | 7 (98.4) | 2 (99.0) | 2 (99.7) | 1 (100.0) | ≤0.03 to >16 | 0.25 | 1 | 42.2 | 54.0 | 3.8 | ||
| Azithromycin | 73 (23.3) | 99 (55.0) | 58 (82.1) | 32 (92.3) | 6 (94.2) | 18 (100.0) | ≤2 to >64 | 4 | 16 | 92.3 | - | 7.7 | ||||||
| Ceftriaxone | 256 (81.8) | 10 (85.0) | 12 (88.2) | 3 (89.8) | 4 (91.1) | 28 (100.0) | ≤0.25 to >8 | ≤0.25 | 4 | 88.8 | 1.0 | 10.2 | ||||||
| Chloramphenicol | 49 (15.7) | 99 (47.3) | 43 (61.0) | 13 (65.2) | 12 (69.0) | 97 (100.0) | ≤2 to >64 | 8 | >64 | 61.0 | 4.2 | 34.8 | ||||||
| Trimethoprim/sulfamethoxazole | 82 (26.2) | 45 (40.8) | 35 (51.8) | 21 (58.5) | 9 (61.3) | 121 (100.0) | ≤0.25 to >8 | 1 | >8 | 58.5 | - | 41.5 | ||||||
| Typhoidal (52) | ||||||||||||||||||
| Ceftolozane/tazobactam | 9 (17.3) | 17 (50.0) | 25 (98.1) | 1 (100.0) | 0 (100.0) | 0 (100.0) | 0 (100.0) | 0 (100.0) | ≤0.12 to 1 | 0.25 | 0.5 | 100.0 | 0.0 | 0.0 | ||||
| Ampicillin | 2 (3.8) | 1 (5.8) | 1 (7.7) | 6 (19.2) | 4 (26.9) | 38 (100.0) | ≤2 to >64 | >64 | >64 | 7.7 | 11.5 | 80.8 | ||||||
| Levofloxacin | 31 (59.6) | 1 (61.5) | 2 (65.4) | 6 (76.9) | 3 (82.7) | 5 (92.3) | 2 (96.2) | 1 (98.1) | 1 (100.0) | 0 (100.0) | ≤0.03 to 8 | ≤0.03 | 1 | 65.4 | 26.9 | 7.7 | ||
| Azithromycin | 2 (3.8) | 14 (30.8) | 25 (78.8) | 11 (100.0) | 0 (100.0) | 0 (100.0) | ≤2 to 16 | 8 | 16 | 100.0 | - | 0.0 | ||||||
| Ceftriaxone | 46 (88.5) | 0 (88.5) | 5 (98.1) | 0 (98.1) | 1 (100.0) | 0 (100.0) | ≤0.25 to 4 | ≤0.25 | 1 | 98.1 | 0.0 | 1.9 | ||||||
| Chloramphenicol | 7 (13.5) | 15 (42.3) | 18 (76.9) | 0 (76.9) | 2 (80.8) | 10 (100.0) | ≤2 to >64 | 8 | >64 | 76.9 | 0.0 | 23.1 | ||||||
| Trimethoprim/sulfamethoxazole | 22 (42.3) | 9 (59.6) | 8 (75.0) | 1 (76.9) | 1 (78.8) | 11 (100.0) | ≤0.25 to >8 | 0.5 | >8 | 76.9 | - | 23.1 | ||||||
| Nontyphoidal (261) | ||||||||||||||||||
| Ceftolozane/tazobactam | 82 (31.4) | 59 (54.0) | 97 (91.2) | 14 (96.6) | 5 (98.5) | 2 (99.2) | 1 (99.6) | 1 (100.0) | ≤0.12 to >16 | 0.25 | 0.5 | 98.5 | 0.8 | 0.8 | ||||
| Ampicillin | 20 (7.7) | 9 (11.1) | 12 (15.7) | 4 (17.2) | 4 (18.8) | 212 (100.0) | ≤2 to >64 | >64 | >64 | 15.7 | 1.5 | 82.8 | ||||||
| Levofloxacin | 60 (23.0) | 8 (26.1) | 30 (37.5) | 93 (73.2) | 46 (90.8) | 16 (96.9) | 5 (98.9) | 1 (99.2) | 1 (99.6) | 1 (100.0) | ≤0.03 to >16 | 0.25 | 0.5 | 37.5 | 59.4 | 3.1 | ||
| Azithromycin | 71 (27.2) | 85 (59.8) | 60 (82.8) | 21 (90.8) | 6 (93.1) | 18 (100.0) | ≤2 to >64 | 4 | 16 | 90.8 | - | 9.2 | ||||||
| Ceftriaxone | 210 (80.5) | 10 (84.3) | 7 (87.0) | 3 (88.1) | 3 (89.3) | 28 (100.0) | ≤0.25 to >8 | ≤0.25 | 8 | 87.0 | 1.1 | 11.9 | ||||||
| Chloramphenicol | 42 (16.1) | 84 (48.3) | 25 (57.9) | 13 (62.8) | 10 (66.7) | 87 (100.0) | ≤2 to >64 | 8 | >64 | 57.9 | 5.0 | 37.2 | ||||||
| Trimethoprim/sulfamethoxazole | 60 (23.0) | 36 (36.8) | 27 (47.1) | 20 (54.8) | 8 (57.9) | 120 (100.0) | ≤0.25 to >8 | 2 | >8 | 54.8 | - | 45.2 | ||||||
Tazobactam was tested at a constant concentration of 4 mg/L, and breakpoints are those for the ceftolozane component; trimethoprim/sulfamethoxazole was tested at a 1:19 ratio of trimethoprim to sulfamethoxazole, and breakpoints are those for the trimethoprim component.
Susceptible breakpoint values are shown in boldface.
Below or equal to the respective MIC.
Above or equal to the respective MIC.
MIC50, concentration required to inhibit the growth of 50% of the isolates tested; MIC90, concentration required to inhibit the growth of 90% of the isolates tested.
According to the Clinical and Laboratory Standards Institute, 2021 (46). S, susceptible; I, intermediate; R, resistant.
TABLE 2.
Antimicrobial activities of ceftolozane/tazobactam and six comparator agents against nontyphoidal Salmonella clinical isolates classified according to serogroup
| Antimicrobial agenta | MIC rangeb | Group B (n = 88) |
Group C (n = 22) |
Group D ( n = 139) |
Group E (n = 7) |
Groups G, I, Q (n = 5) |
|||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC50/90b | % Suscc | MIC50/90b | % Suscc | MIC50/90b | % Suscc | MIC50b,d | % Suscc | MIC50b,d | % Suscc | ||
| Ceftolozane/tazobactam | ≤0.12 to >16 | 0.5/1 | 97.8 | 0.5/0.5 | 95.5 | 0.25/0.5 | 100.0 | 0.5 | 85.7 | 0.25 | 100.0 |
| Ampicillin | ≤2 to >64 | >64/>64 | 12.5 | >64/>64 | 9.1 | >64/>64 | 18.0 | >64 | 0.0 | >64 | 60.0 |
| Levofloxacin | ≤0.03 to >16 | 0.5/1 | 43.2 | 0.06/1 | 59.1 | 0.25/0.5 | 28.1 | ≤0.03 | 71.4 | 0.12 | 60.0 |
| Azithromycin | ≤2 to >64 | 4/>64 | 86.4 | 8/16 | 100.0 | 4/16 | 92.8 | 8 | 71.4 | 4 | 100.0 |
| Ceftriaxone | ≤0.25 to >8 | ≤0.25/>8 | 78.4 | ≤0.25/≤0.25 | 95.5 | ≤0.25/≤0.25 | 91.4 | 0.5 | 71.4 | ≤0.25 | 100.0 |
| Chloramphenicol | ≤2 to >64 | >64/>64 | 36.4 | 8/>64 | 63.6 | 4/>64 | 71.2 | >64 | 42.9 | 8 | 60.0 |
| Trimethoprim/sulfamethoxazole | ≤0.25 to >8 | >8/>8 | 38.6 | >8/>8 | 31.8 | 1/>8 | 69.8 | >8 | 28.6 | >8 | 60.0 |
Tazobactam was tested at a constant concentration of 4 mg/L, and breakpoints are those for the ceftolozane component; trimethoprim/sulfamethoxazole was tested at a 1:19 ratio of trimethoprim to sulfamethoxazole, and breakpoints are those for the trimethoprim component.
MICs are expressed in milligrams per liter. MIC50/90, concentration required to inhibit the growth of 50% and 90% of the isolates tested, respectively.
Percentage of isolates that were susceptible according to the Clinical and Laboratory Standards Institute, 2021 (46).
No MIC90 value is available for these serogroups, because they had fewer than 10 isolates.
TABLE 3.
Antimicrobial activities of ceftolozane/tazobactam and six comparator agents against Salmonella clinical isolates with an ESBL phenotype
| Antimicrobial agenta | MIC (mg/L) |
Susceptibilityc (%) |
||||
|---|---|---|---|---|---|---|
| Range | 50%b | 90%b | S | I | R | |
| Ceftolozane/tazobactam | 0.5 to >16 | 1 | 2 | 100.0 | 0.0 | 0.0 |
| Ampicillin | >64 | >64 | >64 | 0.0 | 0.0 | 100.0 |
| Levofloxacin | 0.06 to 2 | 0.5 | 1 | 20.0 | 70.0 | 10.0 |
| Azithromycin | ≤2 to >64 | 4 | >64 | 70.0 | 30.0 | |
| Ceftriaxone | >8 | >8 | >8 | 0.0 | 0.0 | 100.0 |
| Chloramphenicol | ≤2 to >64 | 8 | >64 | 60.0 | 0.0 | 40.0 |
| Trimethoprim/sulfamethoxazole | ≤0.25 to >8 | >8 | >8 | 40.0 | 60.0 | |
Tazobactam was tested at a constant concentration of 4 mg/L, and breakpoints are those for the ceftolozane component; trimethoprim/sulfamethoxazole was tested at a 1:19 ratio of trimethoprim to sulfamethoxazole, and breakpoints are those for the trimethoprim component.
MIC50, concentration required to inhibit the growth of 50% of the isolates tested; MIC90, concentration required to inhibit the growth of 90% of the isolates tested.
According to the Clinical and Laboratory Standards Institute, 2021 (46). S, susceptible; I, intermediate; R, resistant.
When the activities of the antimicrobial agents against nontyphoidal Salmonella isolates were compared according to serogroup (Table 2), ceftolozane/tazobactam had the highest activity (100%) against serogroup D, G, I, and Q isolates. In contrast, the lowest activity (85.7%) was observed against serogroup E isolates. Moreover, the susceptibilities of serogroup E isolates to nearly all the comparator agents—ampicillin (0.0%), azithromycin (71.4%), ceftriaxone (71.4%), and trimethoprim/sulfamethoxazole (28.6%)—were lower than those of other Salmonella serogroups.
ESBL-producing Salmonella isolates.
All ESBL-producing Salmonella isolates were susceptible to ceftolozane/tazobactam (Table 3), albeit with MIC50/90 values higher (1/2 mg/L) than those of non-ESBL producers (0.25/0.5 mg/L). Azithromycin (70%) was the second most active agent against all ESBL producers. On the other hand, 80% of these isolates were resistant to levofloxacin, and 100% exhibited resistance to ampicillin and ceftriaxone.
Ceftolozane/tazobactam-resistant Salmonella isolates.
Two nontyphoidal Salmonella isolates (S. Typhimurium PW4929 and S. Anatum PW5175) were resistant to ceftolozane/tazobactam, and two nontyphoidal Salmonella isolates (S. Rissen PW5272 and S. Typhimurium PW5304) had intermediate resistance. Their antimicrobial susceptibility profiles are summarized in Table S1 in the supplemental material. Previous studies suggested that the presence of ampC may affect the susceptibility of an isolate to ceftolozane/tazobactam (15, 16). Thus, we investigated whether our ceftolozane/tazobactam-resistant isolates also carried an ampC gene. The resistant isolates PW4929 and PW5175, but not PW5272 and PW5304, were phenotypically confirmed as AmpC-producing isolates by the cefoxitin (FOX) disk diffusion test. PCR amplification further confirmed that PW4929 carried the ampC gene blaCMY-2, while PW5175 carried blaCMY-2 and blaDHA-1.
DISCUSSION
In this study, we showed that ceftolozane/tazobactam is active against most strains of both typhoidal and nontyphoidal salmonellae. Before 1990, the first-line antimicrobials for treating salmonellosis were ampicillin, cotrimoxazole, and chloramphenicol. However, frequent use of these classical first-line drugs resulted in a global spread of multidrug-resistant (MDR; coresistant to ampicillin, cotrimoxazole, and chloramphenicol) Salmonella strains. For instance, in the United States, the incidence of MDR S. Typhimurium isolates rose from 0% before 1960 to 23.2% after 1989 (17). In North India, the incidence of MDR Salmonella strains increased from 34% in 1999 to 66% in 2005 (18). This led to a change to the use of fluoroquinolones for the treatment of salmonellosis, and as a result, fluoroquinolone-resistant salmonellae emerged. As observed in South Asia, the incidence of fluoroquinolone-resistant S. Typhi increased from 2% in 1990 to 81% in 2015 (19). Outbreaks of fluoroquinolone-resistant Salmonella infections were also recognized globally (20–25). This was a cause for concern, since nalidixic acid-resistant Salmonella strains are isolated from blood more commonly than susceptible isolates and might be associated with increased disease severity (26). Third-generation cephalosporins, such as ceftriaxone and cefotaxime, and azithromycin are now the mainstay of treatment for Salmonella infections. However, due to the increasing reliance on these agents, cases of ceftriaxone- or azithromycin-resistant Salmonella strains are already on the rise, with steady reports of these resistant strains around the world (27–33). As a result of the need to search for new antibiotics for the treatment of salmonellosis, we previously performed a preliminary study using Etest, which showed that ceftolozane/tazobactam may be useful against S. Typhi (14). In the present study, we further demonstrated high rates of susceptibility to ceftolozane/tazobactam for both typhoidal (100%) and nontyphoidal (98.5%) Salmonella strains, with a low overall MIC of 0.25/0.5 mg/L relative to those of all the comparator agents, by use of broth microdilution (Table 1). Ceftolozane/tazobactam was also very effective (85.7 to 100% of isolates susceptible) against most of the serogroups of nontyphoidal Salmonella isolates tested (Table 2). In our locality, most nontyphoidal Salmonella strains were locally acquired, whereas more than half of the cases of S. Typhi infections were from other Asian countries, such as India, Pakistan, and Nepal. Based on these in vitro susceptibility results for the Salmonella isolates in this study, in which the strains were collected over the past 10 years, ceftolozane/tazobactam is suggested to be a useful alternative for the treatment of both typhoidal and nontyphoidal salmonellosis.
In addition to being active against salmonellae in general, ceftolozane/tazobactam is also active against ESBL-producing salmonellae. Since their first discovery in 1979 (34), ESBLs have been increasingly found in Gram-negative bacteria, such as E. coli and K. pneumoniae. The presence of ESBL confers resistance to many commonly used β-lactam antibiotics, including the first-, second-, and third-generation cephalosporins. Therefore, an increasing prevalence of ESBL producers has become a serious global health burden. Genetically, CTX-M enzymes have become the most common ESBLs in both the hospital and the community setting in recent years (35). The first ESBL-producing Salmonella strain reported was identified in 1988 as a nontyphoidal Salmonella isolate carrying the SHV-2 gene (36). However, a significant increase in CTX-M-type ESBLs has been detected subsequently in numerous outbreaks of salmonellosis (37–41). ESBL in salmonellae is particularly important, because ceftriaxone is currently one of the most widely used antibiotics for the treatment of salmonellosis. Notably, all the ESBL-producing salmonellae identified in this study carried the CTX-M gene, and correspondingly, all were resistant to ceftriaxone (Table 3). Although ceftolozane is a cephalosporin, the addition of tazobactam, a β-lactamase inhibitor, protects it from hydrolysis by ESBLs. Since ceftolozane/tazobactam has been shown to be very useful for ESBL-producing Enterobacterales such as E. coli and K. pneumoniae (7, 8, 10), we specifically examined its activity against ESBL-producing Salmonella isolates. The results showed that all ESBL-producing Salmonella isolates tested in this study were susceptible to ceftolozane/tazobactam (Table 3), suggesting that this would be a potential alternative treatment for infections caused by ESBL-producing salmonellae.
Conversely, four nontyphoidal Salmonella strains were observed to be resistant, or to have intermediate resistance, to ceftolozane/tazobactam (Table 1; also Table S1 in the supplemental material). The two resistant strains were confirmed to be AmpC producers, carrying either the ampC gene blaCMY-2 alone or both blaCMY-2 and blaDHA-1. AmpC β-lactamases are clinically important hydrolyzing enzymes that mediate resistance to broad-spectrum β-lactams. In many bacteria, the expression of AmpC enzymes is low but is inducible in response to β-lactam exposure, and in some cases, mutations may even result in AmpC hyperinducibility or constitutive hyperproduction (42). Moreover, studies have shown that clinical resistance to ceftolozane/tazobactam can be generated by an accumulation of mutations that lead to overproduction or structural modifications of AmpC (43–45). For example, specific mutations in the ampC gene of MDR P. aeruginosa generate sequence variants named Pseudomonas-derived cephalosporinases, which have impaired hydrolysis of substrates such as penicillins and carbapenems and increased resistance to ceftolozane/tazobactam (43). P. aeruginosa mutant strains with one to four mutations in the conserved residues of ampC also showed increased resistance to ceftolozane/tazobactam (45). Although the activity of AmpC has been studied extensively in some organisms, such as E. coli, P. aeruginosa, and Acinetobacter baumannii, there is little information for Salmonella. In this study, we observe that ampC genes are present in Salmonella strains that have strong resistance to ceftolozane/tazobactam. It is unclear whether induction of AmpC or mutations in the ampC gene are responsible for the resistant phenotype. Further studies with a larger number of AmpC-producing Salmonella strains will be needed to confirm this observation.
Bacteria that are resistant to multiple antibiotics should be examined for their susceptibilities to ceftolozane/tazobactam and other antibiotics on the horizon. In the past 3 decades, antibiotic resistance has become a great concern globally. In 2019, the CDC listed a number of drug-resistant bacteria for which new antibiotics that can potentially be used for treatment are urgently sought. These included Clostridium difficile, carbapenem-resistant Enterobacterales, Neisseria gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, ESBL-producing Enterobacterales, vancomycin-resistant Enterococcus, multidrug-resistant P. aeruginosa, drug-resistant nontyphoidal Salmonella, drug-resistant S. Typhi, drug-resistant Shigella, methicillin-resistant Staphylococcus aureus, drug-resistant Streptococcus pneumoniae, and drug-resistant Mycobacterium tuberculosis. Previous reports have shown that ceftolozane/tazobactam is active against most strains of ESBL-producing Enterobacterales and MDR P. aeruginosa (7–12). In this study, we have shown that ceftolozane/tazobactam is active against most strains of both nontyphoidal and typhoidal Salmonella, including ESBL-producing strains. Therefore, the activities of ceftolozane/tazobactam against other groups of drug-resistant bacteria should also be examined in order to look for alternatives for the treatment of these infections.
MATERIALS AND METHODS
Bacterial isolates.
A total of 313 nonduplicate typhoidal (n = 52) and nontyphoidal (n = 261) Salmonella clinical isolates were prospectively collected from three hospitals in Hong Kong during the period from January 2011 to July 2020. Isolates were derived from routine clinical stool samples (n = 285) or blood culture (n = 28). The species identification of all isolates was confirmed by standard laboratory methods, including matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonik, Bremen, Germany), and they were serotyped by the agglutination test (Oxoid, Thermo Fisher, UK). All isolates were stored at −80°C until use. Ethics approval for this study was provided by the Institutional Review Board of The University of Hong Kong/Hospital Authority.
Antimicrobial agent preparation.
The following antimicrobial agents (with their respective testing concentration ranges given in parentheses) were studied: ceftolozane/tazobactam (0.12/4 to 16/4 mg/L), ampicillin (2 to 64 mg/L), levofloxacin (0.03 to 16 mg/L), azithromycin (2 to 64 mg/L), ceftriaxone (0.25 to 8 mg/L), chloramphenicol (2 to 64 mg/L), and trimethoprim/sulfamethoxazole (0.25/4.75 to 8/152 mg/L). All antimicrobial agents were purchased from Sigma-Aldrich (USA), except for ceftolozane/tazobactam, which was provided by Merck & Co., Inc. (USA). Stock solutions of each antimicrobial agent were prepared according to the manufacturer’s instructions. Prepared stock solutions were aliquoted and stored at −80°C until use.
Detection of ESBL-producing Salmonella isolates.
ESBL-producing Salmonella isolates were detected using the inhibition zone enhancement test, and the results were interpreted according to the CLSI criteria recommended for Enterobacterales (46). Briefly, a 0.5 McFarland standard suspension of overnight culture was inoculated onto Mueller-Hinton agar (Bio-Rad, USA), and antibiotic disks (BD BBL Sensi-Disc; Becton Dickinson, USA) of cefotaxime (CTX; 30 μg) and ceftazidime (CAZ; 30 μg), alone or in combination with clavulanic acid (CLA; 10 μg), were applied by following the standard protocol. The plates were incubated at 37°C for 18 h, and the diameter of the inhibition zone around each disk was measured by following CLSI guidelines. An enhancement of the diameter of the zone of inhibition by ≥5 mm for CTX-CLA or CAZ-CLA over that of the respective antimicrobial disk alone indicated an ESBL phenotype. The quality control (QC) strains E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive controls, respectively.
PCR detection and sequencing of ESBL-encoding genes.
Bacterial DNA was extracted from all ESBL-positive Salmonella isolates by heating one colony in 100 μL distilled water at 95°C for 10 min, followed by centrifugation of the cell suspension. The extracted DNA was then subjected to multiplex PCR assays under the conditions described by Dallenne et al. (47) for the detection of TEM, SHV, OXA-1-like, and CTX-M genes. The mixtures were amplified for 30 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 1 min, with a final extension at 72°C for 7 min, in an automated thermal cycler (Applied Biosystems, USA). Standard precautions were taken to avoid PCR contamination, and no false-positive result was observed for negative controls.
To identify the β-lactamase genes detected, amplified PCR products were purified using the QIAquick gel extraction kit (Qiagen, Germany). Bidirectional sequencing was performed using the primers described by Dallenne et al. (47). The sequences were then compared with known β-lactamase genes in the GenBank database.
Detection of AmpC-producing Salmonella isolates.
AmpC-producing Salmonella isolates were detected using cefoxitin (FOX; 30 μg) disks as described by Polsfuss et al. (48), and the results were interpreted according to CLSI guidelines (46). Briefly, a 0.5 McFarland standard suspension of overnight culture was inoculated onto Mueller-Hinton agar (Bio-Rad, USA), and cefoxitin disks (BD BBL Sensi-Disc; Becton Dickinson, USA) were applied by following the standard protocol. The plates were incubated at 37°C for 18 h, and isolates with a cefoxitin zone diameter of ≤18 mm were considered positive for an AmpC phenotype. The QC strain E. coli ATCC 25922 was included as a positive control.
PCR detection and sequencing of ampC β-lactamase genes.
Bacterial DNA was extracted from Salmonella isolates by heating one colony in 100 μL distilled water at 95°C for 10 min, followed by centrifugation of the cell suspension. The extracted DNA was then subjected to multiplex PCR assays under the conditions described by Pérez-Pérez and Hanson (49) for the detection of ACC, CMY, DHA, FOX, and MOX genes. The mixtures were amplified for 25 cycles of 94°C for 30 s, 64°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 7 min, in an automated thermal cycler (Applied Biosystems, USA). Standard precautions were taken to avoid PCR contamination, and no false-positive result was observed for negative controls.
To identify the β-lactamase genes detected, amplified PCR products were purified using the QIAquick gel extraction kit (Qiagen, Germany). Bidirectional sequencing was performed using the amplification primers. The sequences were then compared with known β-lactamase genes in the GenBank database.
Antimicrobial susceptibility testing.
In vitro antimicrobial susceptibility testing was performed in triplicate by using the broth microdilution method according to CLSI guidelines (50). Ninety-six-well microtiter panels, freshly prepared in-house, were used to test the antimicrobial agents at their respective concentration ranges. The MIC of an antimicrobial agent was defined as the lowest concentration that inhibited visible growth of the microorganism, and results were interpreted as susceptible, intermediate, or resistant according to the CLSI MIC breakpoints recommended for Enterobacterales (46). E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as the QC strains for ceftolozane/tazobactam, and Staphylococcus aureus ATCC 29213 was used as the QC strain for ampicillin, levofloxacin, azithromycin, ceftriaxone, chloramphenicol, and cotrimoxazole.
ACKNOWLEDGMENTS
This work is partly supported by funding from Merck & Co. Incorporated and the Hong Kong Health and Medical Research Fund (HMRF no. 21200512).
Patrick C. Y. Woo has provided scientific advisory/laboratory services to Gilead Sciences, Incorporated, International Health Management Associates, Incorporated/Pfizer, Incorporated, and Merck & Co., Incorporated. Jade L. L. Teng has provided scientific advisory/laboratory services to Pfizer, Incorporated, and Merck & Co., Incorporated. The other authors report no conflicts of interest. The funding sources had no role in study design, data collection, analysis, interpretation, or the writing of the report. The authors alone are responsible for the content and the writing of the article.
Footnotes
Supplemental material is available online only.
Contributor Information
Susanna K. P. Lau, Email: skplau@hku.hk.
Patrick C. Y. Woo, Email: pcywoo@hku.hk.
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