Effective in Vitro Synergy of Piperacillin/Tazobactam Plus either Netilmicin or Aztreonam against Metallo-β-lactamase-producing Pseudomonas Aeruginosa

This study was designed to compare the activity of piperacillin/tazobactam (TZP) in combination with either netilmicin (NET) or aztreonam (ATM) against metallo-β lactamases (MβL) Pseudomonas aeruginosa. Out of 46 nosocomial Ps. aeruginosa isolates, 38 (82.6%) isolates were carbapenem-resistant by disc diffusion susceptibility testing. These were then screened by imipenem + EDTA combined disc test for MβL production. Thirty (78.9%) MβL-producing isolates detected were further tested for antibiotic synergy against NET-TZP and ATM-TZP combinations by Etest synergy and Etest/agar dilution tests. Neither combination showed antagonism. The synergistic effect of NET-TZP combination was detected in [27 (90%) and 28 (93.3%), respectively] and ATM-TZP combination in [24 (80%) and 25 (83.3%), respectively] of the tested isolates by Etest synergy and Etest/agar dilution tests, respectively. Thus, we have determined the synergistic effect of these two combinations for treating MβL-producing Ps. aeruginosa. Significance and Impact of the Study: The present study has shown valuable information on the combination treatment of Ps. aeruginosa and recommends their use for treatment of resistant Ps. aeruginosa.


Introduction
Pseudomonas aeruginosa is an important opportunistic pathogen that plays a serious role in many infections especially in immuno-compromised patients [1]. Infections by this organism are characterized as severe and resistant to treatment with drugs [2]. Metallo-β-lactamase (MβL) production by Ps. aeruginosa is a major challenge because it confers multidrug resistance (MDR) to β-lactam agents, as well as co-resistance to fluoroquinolones and aminoglycosides [3]. Continued emergence and rapid spread of MDR Ps. aeruginosa strains have resulted in increased morbidity, mortality and cost [1,4]. This increased the need for alternative strategies such as combination of anti-pseudomonal antibiotics as an alternative strategy in the management of these cases.
Although aminoglycosides are among the oldest antibiotics to treat serious infections, they have severe side effects such as nephrotoxicity, ototoxicity (vestibular and auditory) and, rarely, neuromuscular blockade and hypersensitivity reactions. They are concentration-dependent, bactericidal agents that have certain limitations including the age of the patient, serum creatinine level and the duration of exposure [5,6]. This and the appearance of new and safer antibiotics resulted in the dramatic drop in the use of aminoglycosides since 1970s. However, their use is being reconsidered again due to the development of MDR among different microorganisms [6]. Netilmicin (NET) is an effective bactericidal drug with a little toxic effect rather than other aminoglycosides which favors its use with severe infections in critical cases that cannot withstand severe toxicity associated with other aminoglycosides [7].
It is well known now that a β-lactam plus an aminoglycoside is the standard for treating severe Ps. aeruginosa infections [8]. This synergy could be explained by the theory that exposure of bacterial cell to β-lactam drugs lessen the barriers, leading to increase the susceptibility to aminoglycosides [9]. The effective in vitro synergistic activity of NET plus β-lactams against resistant Ps. aeruginosa has been documented by several studies with variable degrees. These combinations included NET plus penicillin [10], piperacillin [11], carbenicillin [12], aztreonam , 3rd generation cephalosporins as cefotaxime, 60 Effective in Vitro Synergy of Piperacillin/Tazobactam Plus either Netilmicin or Aztreonam against Metallo-β-lactamase-producing Pseudomonas Aeruginosa cetazidime, cetriaxone and cefoperazone [14].Some of these combinations showed better results than gentamicin when combined with the same β-lactams [11,12,13].
On the contrary, aztreonam (ATM) is a monobactam which has a strong activity against Gram-negative rods with no remarkable toxicity [8,14]. It may be similar in efficacy to aminoglycosides as gentamicin and tobramycin. It could be an effective alternative to aminoglycosides in serious Gram-negative infections [15].
In certain conditions, such as severe infection in patients with poor renal functions or immuno-compromised patients who need prolonged courses of treatment, it is preferable to use dual β-lactam drugs rather than β-lactamaminoglcoside combination. The mechanism of synergy between β-lacatams includes the action of different particles at different penicillin binding proteins and subsequent interference of cell wall synthesis. Another mechanism is the synergistic effect of the inhibition by β-lactamases [18].
The goal of this study was to compare the activity of TZP in combination with either NET or ATM against MβL-producing Ps. aeruginosa using two methods; Etest synergy and Etest/agar dilution.

Bacterial Isolates and Growth Conditions:
This study included 46 nosocomial Ps. aeruginosa isolates obtained from Microbiology Laboratory of Infection Control Unit. Pure cultures were preserved on 15% glycerol in screw top tubes at -70º C until use. Tubes were transported immediately to the Microbiology Laboratory of Microbiology and Immunology Department, Faculty of Medicine for further processing. To recover the bacteria: the frozen surface of the culture was scraped with a sterile inoculating loop, the bacteria that adhere to the loop were streaked onto the surface of plates and the frozen culture was returned to storage at -70 °C [19]. Blood agar and MacConkey's agar plates were used for subculture with incubation at 37°C for 24 hours. Any growth was identified as Ps. aeruginosa by conventional microbiology methods; colony morphology, Gram film and biochemical reactions [20].
Isolates Data were collected retrospectively from patients fulfilling criteria of Centre for Disease Control (CDC) for nosocomial infections [21] and admitted between January 2015 to July 2015 in Tanta University Hospitals, Egypt. These 46 nosocomial Ps. aeruginosa isolates were collected from patients admitted to emergency ICU , pediatric ICU and surgery wards (18 , 17and 11 respectively).

Screening Test for MβL:
Ps. aeruginosa isolates resistant to imipenem or meropenem or both were subjected to the screening test for MβL production by imipenem (Oxoid, Uk) + EDTA (Zhonglan Industry Co, China) combined disc test as described previously by Yong et al . [23]. Isolates with enhancement of zone size of ≥ 7 mm between imipenem + EDTA disc compared with imipenem disc alone were considered as MβL-positive. One Ps. aeruginosa strain producing IMP and another one producing VIM that were confirmed by PCR and provided by our laboratory, were used as MBL-positive controls [24]. Ps. aeruginosa ATCC 27853 was used as a MβL -negative control.

Antibiotic Synergy Testing
In this study, we chose to use the Etest synergy to measure the in vitro synergistic activity between antibiotic combinations. It is an easy and rapid method with low cost, and it has a high agreement with the time-kill assay test [25,26]. In addition, Etest/agar dilution was used to confirm results obtained by the Etest synergy technique [27,28]. NET (Merck Schering Plough, Nestpan-Cipla, USA) was incorporated into MHA at concentration of 0.06 mg/L as previously described [22,29]. Plates were inoculated with 0.5 McFarland suspensions of each isolate, then TZP Etest strips were applied. After 24hrs incubation at 37°C, the strips were read and the Etest MICs were compared to a series performed in the absence of TZP. The same was done with ATM (Bristol Mayers squibb, Princeton, USA) using concentration of 0.5 mg/L and then TZP Etest strips were applied. In this method, we used the least concentration of the drug to be added to the MHA in case of Ps. aeruginosa [22,29].
The FIC was calculated for each antibiotic in each combination by using the following formula: Synergy was defined by a Ʃ FIC of ≤0.5. Antagonism was defined by a Ʃ FIC of >4. Interactions represented by a Ʃ FIC of Ʃ 0.5 but ≤4 were termed indifferent.
aeruginosa ATCC 27853 was used as control strain.

Ethical Consideration
It was approved by the Ethical Committee of the Faculty of Medicine, Tanta University, Egypt.

Statistical Analysis
Data were analyzed using statistical package of social science (SPSS) version 21. Qualitative data were expressed as frequency and percentage. Chi-square test was used to examine the relation between qualitative variables. P-value less than 0.05 was considered significant.
By disc diffusion method, all MβL isolates were sensitive for both colistin and polymyxin B, while (23.3%, 10%) were sensitive for ATM and NET, respectively. Intermediate sensitivity was (43.3%) for piperacillin and (33.3%) for both TZP and ATM. Absolute resistance (100%) was found with all other tested antibiotics ( Table 2). These results identified our choices for the preferable combinations to be tested.

Discussion
Infection with Ps. aeruginosa is a very serious problem due to difficulties associated with treatment options for resistant isolates and poor response rates of patients. In addition, it is usually recruited from immuno-compromised patients with nosocomial infections. Therefore, special care should be taken while determining its antibiotic susceptibility pattern and prescribing the suitable antibiotic therapy. During the last decades, many resistance enzymes had been detected to be produced by Ps. aeruginosa, including Extended-spectrum β-lactamases (ESBL), AmpC and MβL. MβL enzymes confer resistance to all β-lactams (except ATM) and usually to aminoglycosides [30]. MβL-producing Ps. aeruginosa is an emerging threat as it can hydrolyze carbapenems which are given as a last resort to the patient having infection with ESBL and AmpC-lactamase producing Ps. aeruginosa [31].
In this study, 30 out of 38 (78.9%) carbapenem resistant Ps. aeruginosa isolates were positive for MβL production by imipenem + EDTA double disk synergy test. This is a relatively high ratio when compared with finding of Abd El-Baky et al [31] where only 31(53.4%) of their 58 Ps. aeruginosa isolates were MβL producers, and that of Asghar study [32] who reported that MβL-producing Ps. aeruginosa were identified in only 76 (15.9%) of their 478 isolates. While a close result was obtained by Toval et al. [33] where their MβL production ratio was 102 (81.6%) out of 125 carbapenem resistant Ps. aeruginosa isolates. This high ratios of carbapenem resistance and MβL-producing Ps. aeruginosa in our institution may be due to absence of antibiotic policy and the misuse of antibiotics especially β-lactams and carbapenems.
According to antibiotic susceptibility pattern, we noticed that all MβL positive Ps. aeruginosa isolates were sensitive to polymyxin B and colistin. All 30 isolates showed absolute resistant (100%) to amikacin, gentamicin, ceftazidime, cefepime and ciprofloxacin. While, number of resistant isolates to NET was 27(90%), TZP 20(66.7%), piperacillin 17(56.7%) and ATM 13(43.3%). These results supported the measurement of the synergistic effect of TZP combined with either NET or ATM on MβL-positive isolates.
TZP is a β-lactam/β-lactamase inhibitor combination that has a broad spectrum activity against most Gram-positive and Gram-negative aerobic and anaerobic bacteria [34]. The addition of tazobactam enhances the antimicrobial activity of piperacillin against most β-lactamase-producing organisms [35]. As regard drug-drug interaction, when aminoglycosides are administered in combination with piperacillin in patients with end-stage renal disease requiring hemodialysis, the concentrations of the aminoglycosides (especially tobramycin) may be significantly altered and should be monitored. Probenecid if administered concomitantly with TZP, it prolongs the half-life of piperacillin by 21% and that of tazobactam by 71%. In addition, coagulation parameters should be monitored regularly during simultaneous administration of high doses of heparin and oral anticoagulants [36]. TZP is generally safe, well tolerated and of low cost. This renders it to be a reliable option when used in combination with other drugs for the early empiric treatment of moderate-to-severe infections [34,37]. On the other hand, NET is an ethyl derivative of dehydrogenated C1a gentamicin and is active against many gentamicin-resistant organisms. It has the least nephrotoxicity and ototoxicity among the other aminoglycosides [38,39]. Although, some studies did not find NET to be useful against gentamicin-resistant Ps. aeruginosa [40,41]. Others like Biswal et al., [42] detected less resistance of Ps. aeruginosa to NET (58.62%) when compared with amikacin and gentamicin (81.03% for each), which is consistent with our findings.
In the current study, synergy was detected between TZP and NET (90-93.3%) by Etest synergy and Etest /agar dilution respectively with no antagonism. Several studies measured the synergy between TZP and aminoglycosides like tobramycin, gentamicin and amikacin. As far as we know, this is the first attempt for detecting the in vitro synergy between TZP and NET as one of the aminoglycosides. An early study was performed on the combination between carbenicillin and NET which detected a synergy rate of (45.65%) in Ps. aeruginosa isolates [40]. Consistent with our study, a clinical trial noted the effectiveness of NET-TZP combination as a safe excellent early empiric therapy for high-risk febrile neutropenia in pediatric cancer patients [43]. Studies on the other aminoglycosides detected synergy between TZP plus either; tobramycin (50%) [44], amikacin (95.9%, 42%) [44,45] respectively , arbekacin (100%) [3] and gentamicin (33.33%) [46].
On the other hand, less synergy was detected between TZP and ATM (80-83.3%) in this study by Etest synergy and Etest /agar dilution respectively, with no antagonism. Fujimura et al. [3] tested this combination in vitro and reported synergy rate (89.9%) with no antagonism, which is similar to our results. Also investigators supported the in vivo and in vitro synergy between ATM and piperacillin alone, even without the tazobactam addition [45,47]. Nevertheless, Pinheiro et al. [48] did not recommend the ATM-TZP combination as the best combination in empirical or definitive treatment against Ps. aeruginosa.
No statistically significant differences in synergy rates were found between the two combinations using both methods (P-value = 0.227 and 0.424). This may refer to the effectiveness of both combinations to overcome MβL-producing Ps. aeruginosa.

Conclusions
This study determines that NET-TZP and ATM-TZP combinations were effective against MβL-producing Ps. aeruginosa. This may confer better choices in empirical therapy, with less toxicity especially in critically ill patients.