Bacteria can evolve resistance to antibiotics. Even without changing genetically, bacteria also can display tolerance to antibiotic treatments. Many antibiotics are also broadly acting, as can result in excessive modifications of body microbiomes. Particularly for antibiotics of last resort or in treating extremely ill patients, antibiotics furthermore can display excessive toxicities. Antibiotics nevertheless remain the standard of care for bacterial infections, and rightly so given their long track records of both antibacterial efficacy and infrequency of severe side effects. Antibiotics do not successfully cure all treated bacterial infections, however, thereby providing a utility to alternative antibacterial approaches. One such approach is the use of bacteriophages, the viruses of bacteria. This nearly 100-year-old bactericidal, anti-infection technology can be effective against antibiotic-resistant or -tolerant bacteria, including bacterial biofilms and persister cells. Ideally phages could be used in combination with standard antibiotics while retaining their anti-bacterial pharmacodynamic activity, this despite antibiotics interfering with aspects of bacterial metabolism that are also required for full phage infection activity. Here I examine the literature of pre-clinical phage-antibiotic combination treatments, with emphasis on antibiotic-susceptible bacterial targets. I review evidence of antibiotic interference with phage infection activity along with its converse: phage antibacterial functioning despite antibiotic presence. Bacteriophages, or phages, are the viruses of bacteria. A majority of bacteriophages display lytic infection cycles. These involve phages infecting bacteria lethally and, in the process, producing new bactericidal agents, i.e., new phage virions. Phages have been employed clinically to treat bacterial infections for roughly one-hundred years ,2, and mGiven this history, an important question arises: Why has phage therapy not caught on more broadly as a reasonable alternative to antibiotics, particularly in North America and much of Europe? This question is especially relevant in light of issues associated with standard antibiotic effectiveness, the need for new approaches to treatment of bacterial infections , and sevAntibiotics are expected to interfere with aspects of bacterial physiology that can be crucial to phage antibacterial activities, e.g., by interfering with bacterial ribosome functioning. Emphasis here therefore is on documenting the impact of antibiotics on phage-infection pharmacodynamic aspects, i.e., retention of phage ability to negatively impact targeted bacteria despite antibiotic co-treatment. These pharmacodynamic properties include a retention, by bacteria-infecting phages, of both gene expression and antibacterial activity, and also of associated in situ—within treated patients—phage virion production. I address in particular the question of whether or to what degree antibiotic treatments may be antagonistic to the activity of phage infections, and this is especially given treatments in which antibiotic concentrations are greater than or equal to MICs (minimum inhibitory concentrations) ,47,48 foA defining characteristic of the literature concerning phage infection activity in the presence of antibiotics, given bacteria presumptive exposure to ≥1× MIC of antibiotic, is that different studies tend to present only one of two more-or-less opposing perspectives. These are those studies providing evidence or at least a premise that antibiotics will tend to profoundly interfere with phage infection outputs, particularly interfere with phage virion production, versus studies which suggest instead that antibiotic treatment of bacteria will not substantially interfere with phage infection outputs, or at least not interfere to a point where phage antibacterial activity is excessively diminished. It is the evidence underlying the bases of these two contradictory pharmacodynamic, phage-associated viewpoints that I review here. I start by looking at evidence consistent with antibiotic interference with phage infection activities and then consider evidence instead for continued phage infection activity despite antibiotic presence. A tentative overall conclusion is that the more complex the treatment environment, such as especially as found in vivo, then potentially the more compatible that phage therapy may be with antibiotic treatment.The bulk of this article serves as a review of pre-clinical experimentation in which phage treatment or phage infection of bacteria is combined with antibiotic treatment. Emphasis is on interpretation of studies especially regarding the potential for phages to display various activities during their infection of bacteria. These activities, in terms of positive phage pharmacodynamic impacts on bacteria, can be differentiated into bactericidal effects, bacteriolytic effects, and also virion production by phage-infected bacteria. Generally, virion production and subsequent release by lytic phages would imply phage-mediated bacterial lysis, which in turn would imply phage-mediated bactericidal effects. Assessing experiments for these effects, as they may occur in the presence of 1× MIC or greater concentrations of antibiotic, however is not always straightforward, as considered in this section.Firstly, various experiments or controls are useful for assessing antibiotic impact on phage infection activities. First are determinations of MICs for bacterial hosts. Conditions used for testing antibiotic impact on phage infection activity ideally would be equivalent to those conditions used for MIC determinations in terms of use of the same medium, use of the same antibiotic-incubation time frames, use of the same bacterial densities, and use of bacteria found in the same physiological state, such as log- versus stationary-phase. Second is comparing phage infection activities during antibiotic treatments to phage infection activities as occur under antibiotic-minus conditions; such antibiotic-minus infections in principle should allow full phage infection activity. Therefore, phage-only, antibiotic-only, phage-plus-antibiotic, and neither phage nor antibiotic treatments ideally would represent the minimum number of comparisons provided by an experiment. Third, if possible, it can be helpful to compare experiments using antibiotic-susceptible bacteria to equivalent experiments involving antibiotic-resistant host mutants. Phage infections of such bacteria may also allow full phage infection activity.Exposure of bacteria to an antibiotic to which a bacterium is susceptible can in principle block subsequent phage infection-mediated bacteria killing. This proposed blockage can be due for example to antibiotic-caused interference with phage gene expression. Either with or without phage-mediated bactericidal activity, however, bacteria in the presence of 1× MIC antibiotic should not produce either colonies or turbid cultures. This has the effect of making it difficult to assess phage infection-mediated bactericidal activity, versus bacteriolytic or phage production activities, particularly while phage-infected bacteria are still in the presence of antibiotic, i.e., as measured in terms of reductions in bacterial colony-forming units (CFUs). Phage bactericidal activity might instead be assessed by first diluting antibiotic away from bacteria so that they may produce colonies if still able to, as following phage adsorption, but this would not be proof that phage-mediated bactericidal activity would also be retained while phage infections are still in the presence of antibiotic.Phage-associated anti-biofilm activity can occur also as a consequence of the presence of biofilm-matrix degrading enzymes ,51. SuchDelays can exist between antibiotic application and the point of full antibiotic impact on bacterial metabolism, thereby potentially allowing for phage infection activity prior to full antibiotic impact on bacteria. Experiments in which phages and antibiotics are applied simultaneously or in which antibiotics are applied minutes after phage application therefore might provide a false sense of the potential for or degree of phage infection activity in the presence of an antibiotic. In addition, not all antibiotics are rapidly acting, in terms of degrading bacterial metabolic activity, e.g., such as cell-wall synthesis-inhibiting antibiotics for which duration of exposure along with antibiotic concentration are important components of their impact . Phage infection activity thus in principle could occur during experiments prior to substantial antibiotic impact on bacterial functioning.resistant bacteria, that is, so long as those bacteria have first been allowed to grow to relatively high densities. Any of these scenarios could result in a false impression of occurrence of phage infection activity, such as virion production in the presence of even seemingly substantially antibiotic-impacted populations of antibiotic-susceptible bacteria. Antibiotic concentrations can decline over the course of experiments, particularly given long (multi-day) treatments and application of only a single antibiotic dose ,48. SuchGenerally, it also is important to be aware that there is a difference between phage activity as occurs immediately upon infection  versus bacterial resistance evolution as occurs over longer time frames. Not all experimental treatments, particularly ones of long duration and that are assessed only in terms of changes in relative bacterial presence, will necessarily distinguish between these two phenomena. Resistant bacteria, especially given phage and antibiotic treatments of long duration, can by their presence mimic reduced bactericidal activity by individual agents as relative to phage-antibiotic combined treatments . This potentially false negative phage-mediated bactericidal activity would be due to different likelihoods of bacterial mutation in combination with resistant-bacterium grow back given application to cultures of single versus multiple antibacterial agents. Comparisons of bacterial numbers following, e.g., 24 or more hours of treatment, thus may be less indicative of phage bactericidal activity in the presence of antibiotic than somewhat sooner assessments, i.e., as made prior to substantial resistant-mutant bacterial grow back.−MOI = 1 – e−1 = 63% of bacteria present. Therefore, any bacteria killing that is in excess of 63%, particularly killing that is attributable to phage action, could in this example be indicative of excessive virion numbers and thereby of in situ phage virion production following dosing [Lastly, phage virion production can be inferred, within more complex experimental systems such as those consisting of biofilms, from measurements of bacteria-killing activity. Doing so requires knowledge of initial ratios of added phages to bacterial numbers and is based on Poisson distributions . For exag dosing . The likAntibacterial agents can be distinguished into those which are bacteriostatic versus those which are bactericidal . A generThat antibiotics might interfere with phage infection activity is not a new idea, e.g., as documented by Adams . Indeed,Ralstonia solanacearum—ΦRSF1, ΦRSL1, ΦRSL2, and ΦRSB1—for which phage virion production is fully blocked given bacterial exposure to 5 μg/mL rifampin (20 min incubation prior to phage infection). Here the calculated MIC for this host was 3 μg/mL, and thus phage virion-production was inhibited given exposure to ~2× MIC. Furthermore, as Tagliaferri et al. [Mycobacterium tuberculosis as host. In this section, I summarize experiments indicating a potential for antibiotics to interfere with such phage infection activities. As follows, CFU stands for Colony-Forming Units and PFU stands for Plaque-Forming Units. As throughout this review, I both summarize the results of individual studies and provide my interpretations of those results, presenting the material in ascending author-date order. See By way of example, regarding antibiotic interference with phage infection activity, Matsui et al.  describei et al.  suggest M. tuberculosis is both an important human pathogen and an extremely slow growing bacterium. As a consequence of the latter property, diagnosis of antibiotic resistance in M. tuberculosis can take far longer than can be clinically desirable. To reduce the duration of this process, such as down to on the order of a few days, a number of groups have explored the use of phages as indicators of M. tuberculosis metabolic activity in the presence of antibiotics. Here reduced bacterial metabolic activity, as a consequence of antibiotic exposure, is assumed to correlate with reduced phage infection activity, activity as measured by various means. In this subsection I review a sampling of this literature, which mostly is supportive of and indeed premised upon the idea that antibiotics can be antagonistic to phage infection activities.Mycobacterium smegmatis phage TM4, with a goal of assessing gene expression during infection of M. tuberculosis in the presence of antibiotic. They found that after 48 h of antibiotic incubation, luciferase expression is knocked out when the treated bacteria are antibiotic susceptible but not when bacteria instead are antibiotic resistant. In this case, the antibiotics were isoniazid (1 μg/mL), rifampin (2 μg/mL), or streptomycin (6 μg/mL). As this was a luciferase-based test, light output would be dependent not just on reporter-gene expression following phage infection but also on ATP levels within the infected bacteria, levels of both which should positively correlate with phage infection activity . A multiplicity of 1000 was employed and 80-fold greater luminescence was reported without either rifampin (2 μg/mL) or streptomycin (6 μg/mL) present, and ten-fold greater without isoniazid (1 μg/mL), thus indicating substantial inhibitions of phage infection activity by these antibiotics. These results were not correlated in this study with MICs, however.A luciferase-encoding reporter phage was developed by Jacobs et al. , based oM. tuberculosis. Phage plaque formation after termination of antibiotic treatment was used as an indicator of phage virion production, with Mycobacterium smegmatis serving as the plaquing indicator bacterial strain. Antibiotic treatments prior to phage addition (~10 9 PFU/mL in situ concentration) consisted of three days of incubation in the presence of isoniazid at concentrations of 2 μg/mL or rifampin at concentrations of 4 μg/mL . Generally, plaque formation was associated, as expected, with bacterial antibiotic resistance while no plaque formation was seen given antibiotic sensitivity, as was found to be true for 44 of 46 isolates tested. The two outlying M. tuberculosis isolates were reportedly rifampin-susceptible but nevertheless able to support phage virion production despite the antibiotic’s presence. Equivalent concordance of potential to support plaque formation, and isoniazid resistance versus sensitivity, was seen for 40 of 46 isolates tested: two otherwise resistant strains failed to support plaque growth in the presence of isoniazid, while four susceptible isolates did support plaque growth. Generally, then, prior antibiotic treatment was found to interfere with the ability of antibiotic-susceptible but not antibiotic-resistant bacteria to support the production of at least one phage virion, though with some exceptions. Of possible relevance to these exceptions, standard MIC determinations were done over one- to two-week periods while antibiotic treatment prior to phage infection was for a total of three days.Wilson et al.  used phaM. tuberculosis isolates with rifampin (5 μg/mL) and then with phage D29 (107 PFU/mL). Bacteria were incubated in the presence of antibiotic for 24 h and then in the presence of phages for 90 min at 37 °C. After inactivation of unadsorbed phages, the production of new phages was detected via spot assays . In theMcNerney  for a foM. tuberculosis exposure to rifampin on phage D29 virion production. In this assay, colorimetrically indicated M. smegmatis culture-clearing (in the absence of antibiotic) was the indicator that phage virion-production had previously occurred in association with overnight rifampin-treated M. tuberculosis. This broth-based M. smegmatis clearing was compared with a plaquing-based determination of virion production, also post antibiotic exposure. In a small fraction of cases, phage virion production was seen given 2 μg/mL rifampin treatment of M. tuberculosis but not at 10 μg/mL. In most cases those bacterial strains deemed rifampin-susceptible, as based on genome sequence information, gave rise to blocked phage virion production even at the lower rifampin density (at least 70 out of 85). In one case, however, phage virion production was seen even at the higher rifampin concentration. As with all of the studies so far described, results predominantly were consistent with a lack of phage infection activity given bacteria exposure to antibiotic. MIC for M. tuberculosis strains were not reported.McNerney et al.  developeM. tuberculosis exposure to several different antibiotics: amikacin, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin, linezolid, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifampin, and streptomycin. As part of this analysis, it was found that treatment with rifampin for 3, 6, 24, or 48 h prior to 24 h of phage treatment yielded substantially less phage signal with rifampin-susceptible versus rifampin-resistant bacteria. Equivalent treatment with isoniazid by contrast revealed a delay in impact. That is, 3- and 6-hour pre-incubations yielded similar phage signal as seen with a multidrug-resistant control, but by 24 and 48 h of antibiotic pre-incubation there was substantially less phage signal.Pholwat et al.  used qPCThe difference in the impacts of the two drugs is accounted for by differences in their modes of action, with one acting bacterial cell-internally—where drug concentrations tend to be particularly relevant (rifampin inhibits RNA polymerase)—and the other acting by inhibiting cell-wall synthesis, where duration of drug exposure is also a key variable . Nevertheless, and contrary to the rest of the results reviewed in this section, this observation of delayed effects with isoniazid is consistent with phage production despite substantial antibiotic pre-treatment of susceptible bacteria, which in turn is suggestive that not all combinations of phage types, antibiotic types, antibiotic concentrations, and durations of pre-incubation will necessarily result in blocks on phage antibacterial activity, even if MICs are exceeded.The antibiotic concentrations used were those provided by the World Health Organization as “critical concentrations”, that is, the amount required to observe therapeutic effects in patients given treatment of drug-susceptible bacteria. Experiments with all of the above-listed antibiotics—given 48-h pre-incubation and 24-h phage exposure—were consistent with a strong concordance between qPCR-based predictions of phage propagation and non-phage-based determinations of drug sensitivity versus resistance (concordance was mostly 100% but was 97% for capreomycin and 82% for ethionamide). These data, as with those seen in previous studies and except given insufficient duration of pre-incubation with isoniazid, are consistent with antibiotic-associated inhibition of phage virion production.M. tuberculosis as a host. Indeed, in 2010 a meta-analysis based on 31 different studies was published on the subject by Minion and Pai [M. tuberculosis as phage hosts versus additional bacterial types, relatively limited phage choice, and also questions concerning bacterial viability following long pre-incubations with antibiotic. Especially the first two of these limitations are addressed in the following two sub-sections.Overall, exploration of inhibition of phage infection activity in the presence of antibiotic, particularly employing the antibiotic, rifampin, has been extensive using  and Pai . LimitatPseudomonas aeruginosa is able to replicate, producing virions more or less normally in the presence of 400 μg/mL of rifampin, which is a concentration of rifampin that completely inhibits the growth of a different phage, LUZ19 [Bacillus subtilis phage PBS2, equivalent results were seen with rifampin treatment  and also with host RNA polymerase-inhibiting streptolydigin and streptovaricin, but not with the DNA-binding, RNA-synthesis inhibiting actinomycin D and lucanthone antibiotics [Because rifampin is an inhibitor of host RNA polymerase, any phages which carry their own RNA polymerase within their virions, deliver this enzyme upon adsorption, and otherwise do not require their host’s RNA polymerase during infections may not be susceptible to rifampin treatments of bacteria. Sensitivity would be in terms of rifampin inhibiting virion production ability, and phage resistance would be seen even when host bacteria are rifampin susceptible. In particular, phage φKZ of e, LUZ19 . Phage φibiotics . MissingB. subtilis fails to lyse or produce new virion particles given exposure to 1 μg/mL rifamycin starting eight min prior to phage addition (MOI = 5). Both lysis and new virion production occurred more or less normally under the same conditions given infection of rifamycin-resistant mutants of the same host. Under these same conditions, the host was found to be unable to synthesize RNA transcripts as measured via incorporation of 3H uridine. Phage replication also could be blocked given a 2-min but not a 6-min delay between phage adsorption and rifamycin addition (phage addition followed by antibiotic addition), implying that sufficient transcription of phage genes had occurred by 6 minutes to allow the phage to carry out the rest of its life cycle. Phage replication thus is highly impacted by levels of rifamycin which also block host RNA synthesis. MICs for either rifamycin-susceptible or -resistant hosts, however, are not provided.Geiduschek and Sklar  found thB. subtilis. These phages were more or less fully inhibited by rifampin (10 μg/mL) when added sooner after the start of phage infection (MOI = 4) but only partially inhibited by rifampin when added later after the start of phage infection. Burst sizes were less than 1% of those without rifampin addition but increased substantially given rifampin addition at 35 min into infection cycles. MICs were not reported.Hemphill et al.  looked aE. coli was treated with 25 μg/mL rifampin. With a rifampin-susceptible host, “phage growth” was reported as being “<10% of normal” in the presence of rifampin, while “normal” phage growth occurred in rifampin’s absence. Of interest, two hosts which are reported as displaying slight growth given exposure to this concentration of rifampin also are indicated as allowing “±” phage growth . Although not explicitly indicated, I assume that it is phage burst size that is being measured as “phage growth”, indicated as determined via single-step growth experiments [Takeda et al.  explorederiments ,91, withE. coli. Absent either drug, burst size was on the order of 200 in the doubly susceptible bacterium (rifampin and streptolydigin) but only 100 in the doubly resistant bacterium, suggesting existence of costs in phage infection activity associated with host antibiotic resistance. With susceptible bacteria, addition of either drug at 100 μg/mL, immediately after phage adsorption, in amounts which appear to exceed MICs, reduced burst sizes to negligible (1% of no treatment), whereas a 10-min delay in drug addition reduced burst sizes instead to 5% of no treatment.Sokolova et al.  exploredE. coli phage T5 under single-step growth conditions. Burst sizes were reduced from 120 without rifampin down to zero with. Little impact was seen with rifampin-resistant hosts. MICs were not reported.Beckman et al.  determinE. coli, applied rifampin (100 μg/mL), and then assayed for phage virion production. This production was “blocked completely” with addition up to 15 min into the induced infection cycle. Inhibition of phage virion production was not observed, however, with a rifampin-resistant bacterial host. MIC for the E. coli host was not determined.Toussaint and Lecocq  induced Pseudomonas BAL-31 and 0.3 μg/mL rifampin. Burst sizes were reduced to less than 1% given simultaneous (time 0) rifampin addition. The Pseudomonas BAL-31 MIC for rifampin is not indicated.Riveros-Moreno  employedBlasdel et al.  found thM. tuberculosis phages. Exceptional were certain phages whose virions appear to carry non-host, otherwise not antibiotic-susceptible RNA polymerases into newly infected bacteria. Otherwise, production of new virions often appears to be completely blocked in the presence of these various drugs. Unfortunately, however, it is difficult to correlate most of these phage infection-inhibition results, and associated antibiotic concentrations, with the MICs of host bacteria. Therefore, the major conclusion is that given sufficient concentrations of antibiotic, then phage infection activity can be blocked by rifampin, but it remains uncertain whether incubation, e.g., with 1× MIC, would result in equivalent interference. In addition, simply phage bactericidal activity was not measured in these experiments, also such as at 1× or greater MIC. That is, it is conceivable that phage antibacterial activities could be retained even at antibiotic concentrations that nevertheless are able to interfere with phage virion production.These various results are consistent with RNA polymerase-inhibiting antibiotics interfering with infections by different non-E. coli CFUs after 60-min of exposure, and thereby presumably exceeded MIC (50 μg/mL), were also able to nearly or fully prevent the production of virions both by double-stranded phages (T2 and T5) and by single-stranded DNA phages (φR and φX174), but did not prevent the production of virions by RNA phages (β or 10). In these experiments, antibiotic was added 7 min after phages, with bacteria lysed by chloroform 60 min after antibiotic addition and prior to plating. The inhibition of phage T2 was, however, reversible given suspension of phage-infected bacteria into antibiotic-free media. Therefore, nalidixic acid appears to only reversibly inhibit phage bactericidal and indeed virion production ability, at least after 1-hour incubations with the antibiotic. Nalidixic acid, like ciprofloxacin, is an inhibitor of DNA gyrase and topoisomerase. Therefore, these experiments suggest a substantial impact of inhibition of DNA gyrase or topoisomerase on phage replication, though with caveats as well as corroborations as presented by Baird et al. [Taketo and Watanabe  reportedd et al. , discussE. coli phage T7 also using 50 μg/mL nalidixic acid and over 95% burst-size reduction with 10 μg/mL. The Baird et al. results for phage T2, however, seem to contradict those of Taketo and Watanabe, with burst size 34% of that without antibiotic. A possible explanation for this discrepancy is that of a need for prior equilibration of the antibiotic with bacterial cells, where a 30-min pre-incubation reduced burst sizes of phage T4—which was impacted identically to that of phage T2 given no pre-incubation—to only 2% of the burst without antibiotic treatment.Baird et al.  adsorbedInconsistently, no such pre-incubation was employed by Taketo and Watanabe. Baird et al. suggest, however, that adsorption by newly released virions—following lysis but prior to chloroform treatment at 60 min—could explain the loss of phage viability in the Taketo and Watanabe experiments as those authors did not dilute their infective centers in the course of their experiments, with naturally released virions therefore likely to have infected new bacteria, and thus may have been killed upon chloroform treatment. This hypothesis was tested and confirmed by Baird et al. In a separate experiment, the burst size of phage λ following induction was 2.5% that of the no-antibiotic control given dilution into 50 μg/mL nalidixic acid 7 min following the start of induction. This result suggests possibly a complete blockage of virion production given sufficient antibiotic pre-incubation. Although the Baird et al. results are not fully consistent with those of Taketo and Watanabe, they nevertheless are suggestive of a blocking, perhaps in full given sufficient pre-incubation with antibiotic, of virion production by at least some phages by the presence of 50 μg/mL nalidixic acid, though MIC was not determined.E. coli or phage ECA2 (107 PFU/mL) population growth while in the presence of chloramphenicol or tetracycline at 1× MIC. They also observed an absence of phage population growth and probably a lack of phage-mediated bacteria killing in the presence of 1× MIC ciprofloxacin. Although these experiments were performed in phosphate-buffered saline, nevertheless phage population growth was substantial (1 log) and bacterial population growth occurred as well (up to 4 log), both in the absence of antibiotic presence. Therefore, the Valério et al. results are consistent with antibiotic interfering with phage infection activity, but possibly some of the observed antibiotic interference with phage infection activity was intensified given the low nutrient conditions that presumably were present during experiments.Valério et al.  observedThe primary take-home message from this subsection are those of Baird et al. , findingGiven that phage bactericidal activity in the presence of antibiotics—antibiotics to which targeted bacteria are susceptible—can be difficult assess , it is nAt the same time, these experiments, as listed in It is perhaps a naïve assumption that—at 1× MIC for a given antibiotic—bacterial physiology will be sufficiently negatively affected that support of phage infection activity will always be fully curtailed. MIC determinations, that is, generally involve macroscopic evidence of bacterial growth after a defined length of incubation, i.e., the formation of turbid broth cultures or colonies on agar after some period of incubation . This blBroth-culture experiments have a utility of relative ease of execution, ready phage access to all bacteria present (assuming sufficient broth mixing), and relatively low physiological heterogeneity among those bacteria present. Ideally, experiments will explore the impact of measured 1× MIC or greater antibiotic concentrations on phage infection activity, with reporting of evidence for or against bactericidal, bacteriolytic, or phage virion production activities. Furthermore, characterizations of phage infection activities, at a minimum, should consist of phage single-step growth-like experiments ,91, i.e.B. subtilis phage SPO1 (MOI = 6) was able to display bacteriolysis and virion production in the presence of nalidixic acid (50 μg/mL), “at concentrations that drastically inhibited B. subtilis DNA synthesis”. Latent periods, however, were approximately twice as long as without antibiotic, and burst sizes were reduced by roughly 30%. Consistent with display of this level of infection activity, the rates of DNA synthesis by antibiotic-treated, phage SPO1-infected bacteria were substantially higher than as seen with nalidixic acid treated, phage-uninfected bacteria: 0% vs 75% reductions at 5 μg/mL, respectively, and 30% vs 95% at 50 μg/mL. Nonetheless, and unfortunately, it is not possible from the data presented to conclude that antibiotic concentrations exceeded 1× MIC for the bacterial host.Perhaps equivalent to observations of rifampin-resistant phages , Gage anB. subtilis phages φ29, SP50, and SP82 displayed burst sizes of 75%, 44%, and 46% that of the no-nalidixic acid controls as well as lysis delays in the presence of 50 μg/mL nalidixic acid. It is possible, though, that this relatively insubstantial reduction in phage virion production in the presence of nalidixic acid is a consequence of delays in equilibration of antibiotic with bacteria, thereby representing less antibiotic impact on phage virion production than might otherwise occur at that concentration. Specifically, Baird et al. also found that phage T4’s burst size infecting E. coli could be reduced from 34% to 2% of no antibiotic treatment given sooner exposure to antibiotic: delays of 5- to 7-min in antibiotic addition relative to phage addition (phages added first) versus 30 min delays in phage addition relative to antibiotic addition (antibiotic added first), respectively.Baird et al.  found, sBaird et al.  also obsB. subtilis. The antibiotic concentrations used was able to “block completely the DNA replication” of that host. As tested, this included 10 μg/mL of the drug, though at least a 10-min incubation was required before DNA replication ceased. An MOI of 1.5 was used and lysis was followed by optical density (OD540). While drops in OD, presumably as phage-induced, were fairly gradual, continuing for over at least a 100-min period, nevertheless with 10 μg/mL nalidixic acid added 10-min prior to phage addition, lysis kinetics were identical to as seen without the antibiotic present, and in the presence of 50 μg/mL nalidixic acid lysis was simply delayed by about 25 min rather than altered in the rate of lysis once begun. Phage SPP1 thus likely remains at least bacteriolytic even given exposure to nalidixic concentrations that are substantially more than that necessary to interfere with bacterial DNA replication. MIC for the host, though, was not reported.Pedrini et al.  considerB. subtilis phage PBS2 (MOI = 5) of approximately 90% in the presence of simultaneously added 50 μg/mL nalidixic acid, but with burst size seemingly further reduced given antibiotic addition 5- or 15-min prior to phage addition. This is suggestive of substantial blocking of phage virion production but also (again) that delays may exist in equilibration of the antibiotic into cells following its addition to cultures. At this concentration of drug, bacterial DNA replication seems to have been blocked in uninfected cells. The same concentration, however, delayed but did not block phage-induced culture lysis, indicating a retention of at least bacteriolytic activity and presumably bactericidal activity as well (and hence the inclusion of this result under evidence of phage-antibiotic compatibility). Burst size, however, appeared to be substantially reduced.Price and Fogt  observedThis observation of presumptive bactericidal and/or bacteriolytic activities makes sense to the extent that phage gene expression, and thereby production of lysis-inducing proteins, is able to persist even if phage DNA replication is blocked. Addition of 20 μg/mL of nalidixic acid, described as “low levels”, by contrast blocked bacterial DNA replication in uninfected bacteria for over 70 min, but did not block bacterial DNA replication for over 90 min. This lower antibiotic concentration was associated with burst size reductions of about 50% given simultaneous phage and antibiotic addition, or greater reductions (~70%) when phages were added 5- or 15-min after antibiotic, though there was no associated delay in lysis. It is uncertain to what degree antibiotic concentrations of 50 μg/mL may have exceeded MIC. These results nevertheless are consistent with retention of at least some phage antibacterial activity despite nalidixic acid presence, and to a degree even retention of virion production by this phage in the presence of 50 μg/mL of this antibiotic.Mycobacterium aurum as host bacterium and infection by phage D29). This is consistent with cell-wall synthesis inhibitors requiring a fair amount of time before they affect bacterial functioning. For the antibiotic ethambutol, the equivalent ratio to MIC was so high that it was not determinable. This drug too is a cell-wall synthesis inhibitor. Dapsone gave about an order of magnitude greater phage inhibitory value (50% reduction in burst size) than the measured MIC. Although dapsone is not a cell-wall synthesis inhibitor, it is an enzyme inhibitor.David et al.  found thFor all other antibiotics tested , ratios were closer to 1, suggesting indeed an inhibition of at least phage virion production at antibiotic concentrations close to MIC. Of these, colistin and streptomycin had the greatest impacts on phage virion production versus bacterial replication, with the ratio to MIC being 0.78 and 0.54, respectively, e.g., as versus 166 for isoniazid. The colistin result is potentially odd, however, given that this antibiotic too acts on the bacterial cell envelope rather than interfering internally, though then again mycobacteria do not have Gram negative cell envelopes, where Gram-negative pathogens and their outer membranes are the normal targets of colistin. These results are clearly indicative nevertheless of a potential—while infecting this bacterium—for this phage to replicate in the presence of certain antibiotics . A caveat, however, is that drugs were not added until after phage adsorption was considered to have occurred, and thus antibiotic was delayed in its addition, potentially resulting in greater phage infection activity than otherwise might have been seen versus, e.g., given pre-incubation of antibiotic.B. subtilis phage, AR9, which displayed incomplete inhibition of phage virion production given infection exposure to rifampin (50 μg/mL). Indeed, in single-step growth experiments an approximately ten-fold reduction in burst size was reported, as representing a total of only about eight virions produced per phage-infected bacterium, though it is not obvious when antibiotic was applied relative to phages. Antibiotic concentrations at 50 μg/mL alone resulted, after 10-min exposure, also in ~100-fold reductions in cell viability. Therefore, the authors suggest that phage AR9 may in fact not be rifampin susceptible, e.g., as potentially due to the phage virion carrying its own RNA polymerase, but with the decrease in phage burst size related instead to cell killing by the antibiotic. This hypothesis would seem to be somewhat consistent with the indicated 1-log drop in pre-burst phage infective centers with rifampin treatment , and this explanation would be rather than due to more specific inhibition by rifampin of phage functioning during infection.Lavysh et al.  studied Inconsistent with the infection death hypothesis, using a ~ten-fold less rifampin-susceptible host, Lavysh et al.  still seR. solanacearum to replicate in the presence of up to 20 μg/mL of rifampin (MIC = 5 μg/mL). At 1× or 2× MIC, resulting titers were reduced by about one log, whereas at 4× MIC, i.e., 20 μg/mL, they were reduced by roughly 2 logs. The authors suggest that these results are an indication that these phages, like phage PBS2 of B. subtilis [P. aeruginosa [R. solanacearum phages—unlike for phage φKZ, for which candidate subunits were identified and for which no bacterial promoters could be identified—it is more difficult to conclude that phages ΦRP12 and ΦRP31 truly carry their own enzyme within virions or instead for some reason are simply in some manner less susceptible to this antibiotic than the other phages tested.Matsui et al.  tested tsubtilis  or phageruginosa , carry tP. aeruginosa with combinations of a phage cocktail (108 PFU/mL) and either ciprofloxacin or meropenem (both at 2.5× MIC), presumably with each added simultaneously. After six hours of exposure, bacterial numbers were reduced by roughly 6 logs in both cases following either phage-only treatment or combined treatment, versus less than 1-log reductions by antibiotic alone, and approximately 1-log increases without treatment. As the calculated MOI was only 1, the 6-log declines in CFUs are indicative of phage virion production during treatments, both with and without antibiotic presence.Oechslin et al.  treated A. baumannii for 16 h in the presence of phage KARL-1 added at various starting MOIs  and various concentrations of the antibiotics ciprofloxacin, colistin, or meropenem . With antibiotics alone there was substantial resulting culture turbidity at all treatment concentrations, though especially with ciprofloxacin there was some increasing reduction in turbidity with increasing antibiotic concentration. With phage-antibiotic combinations there generally was less turbidity at the end of experiments than with antibiotic alone, even starting with higher antibiotic concentrations  or lower phage MOIs (down to 10−7). Even if differences in end-point turbidities were a consequence of increased likelihoods of mutation to resistance without combination therapy versus with, these results are still suggestive of phage population growth in the presence of antibiotic since phage concentrations would need to exceed MOI = 1 for these combination treatment effects to be observed. What is uncertain, however, is whether this proposed phage population growth occurred in association with antibiotic-susceptible versus antibiotic-resistant bacteria. Their Figure S5, however, is at least suggestive that this latter hypothesis is not correct. Instead, it would appear that experimental conditions might have differed from those that were used to determine MICs such that antibiotic-susceptible bacteria nevertheless were able to at least increase in turbidity over time in the presence of all antibiotic concentrations tested, though mostly with less of an increase than as observed with the untreated control culture . The lower impact, i.e., higher resulting culture turbidities that were observed with the lowest starting phage MOIs (in combination with treatment with ciprofloxacin or colistin), seems to be due to delays in phage population growth to MOIs of greater than 1, that is, as associated with lower phage starting numbers, thereby resulting in less efficient culture clearing.Jansen et al.  followedE. coli. In the same experiments, phage impact on the E. coli target in combination with ciprofloxacin was greater, by nearly 1 log, than ciprofloxacin impact alone, though phage-antibiotic combination treatments reduced bacterial numbers somewhat less than phage impact alone. As starting phage titers were only about 106 PFU/mL, these results, in combination, are consistent with the occurrence of some phage virion production in the presence of ciprofloxacin, at least at 1× MIC.Lopes et al.  found evNo increase in phage numbers was seen with 2× MIC ciprofloxacin, and this was versus an approximately 3-log increase in phage numbers without ciprofloxacin present. No difference in terms of impact on bacterial presence was observed with ciprofloxacin alone versus with phages present as well, also given antibiotic concentrations of 2× MIC. Therefore, overall, at least a small amount of phage virion production appears to be present at 1× MIC ciprofloxacin in this system, but not at 2× MIC.The results presented in this subsection are consistent with at least some phage infection activity in broth bacterial cultures, and specifically phage virion production at antibiotic concentrations of at least 1× MIC. Evidence is also provided that positive phage infection-activity results can be reduced given longer pre-incubations of antibiotic with bacteria prior to phage application. Furthermore, while bacteriolytic activity can be retained in the presence of antibiotic, burst sizes also can be reduced and latent periods extended. Different phages also appear to display different levels of sensitivity to antibiotic presence. In my opinion, the primary take-home messages of this subsection are that antibiotics at concentrations that inhibit bacterial replication do not necessarily also fully interfere with phage infection activity, including in terms of virion production, but that it is important to confirm that this result is retained even given delays between antibiotic addition and subsequent phage addition.A number of studies have been recently published that employ combinations of phages and antibiotics to treat in vitro-grown bacterial biofilms. Interpretation of results of these experiments, in terms of phage-antibiotic interactions and resulting pharmacodynamics, can however be challenging due to the possible heterogeneity of these cultures in terms of the susceptibility of individual bacteria to phages, antibiotic, or both. In addition is the issue of the potential for phages to carry biofilm-matrix degrading enzymes including in association with virions . BiofilmK. pneumoniae one-day-old biofilms—in terms of reductions in numbers of CFUs—than with either phage or antibiotic treatment alone (the name of the phage used does not appear to have been specified by the authors). This result is suggestive of retention of phage anti-biofilm activity despite antibiotic presence. Given that phage doses were of only MOI = 0.01, this result is consistent also with phage virion production in association with the treated biofilm. Note however the potential for heterogeneity in bacterial physiology or in antibiotic susceptibility within biofilms, or indeed the potential for antibiotic to decay over time, which makes it difficult to tell whether the individual bacteria supporting such phage virion production were also bacteria that at the same time were highly affected by antibiotic. Furthermore, amoxicillin as a cell-wall synthesis-inhibiting antibiotic potentially allows phage infection until bacteria either have been killed or nearly killed by amoxicillin activity.Bedi et al.  found so−5 mutations per cell division versus 10−10). As a result, differences in numbers of CFUs between antibiotic-only and phage-plus-antibiotic treatments, particularly for experiments done within relatively small volumes, could be due solely to regrowth of antibiotic-resistant bacteria  but no regrowth of bacteria that are both phage and antibiotic resistant . In other words, differences in numbers of CFUs at the point of bacterial enumeration could be due to one treatment having a greater potential for biofilm elimination to be reversed over time (because of growth of resistant bacteria), and this is rather than due to differences in more immediate pharmacodynamic antibacterial impacts.A concern with experiments in which treatments take place overnight or longer is the potential for regrowth of biofilm by resistant bacteria . That isPhage population growth and therefore virion production nevertheless still must have occurred in the Bedi et al.  experimeK. pneumoniae biofilms with a combination of phage KPO1K2 (MOI = 1) and ciprofloxacin . Ciprofloxacin after 0.5 to 3 h of treatment reduced biofilm CFUs by roughly 1 log. This reduction, however, is relative to the untreated control rather than to bacterial densities that were present at the start of treatments. Ciprofloxacin at this density thus likely was preventing biofilm growth, but otherwise may not have actually reduced bacterial numbers. Reductions associated with phage treatment were roughly 3 to 4 log relative to the bacterial concentrations at the start of treatment, and these amounts were regardless of whether antibiotic was present. Such levels of reduction would appear to be consistent not only with display by this phage of bactericidal activity in the presence of antibiotic, but with phage virion production as well, i.e., as an MOI of 1 alone is insufficient to result in multi-log reductions in bacterial densities.Verma et al.  treated An alternative hypothesis is that phage presence resulted in reduced resistance by biofilm to removal during washing, with washing preceding enumeration of biofilm bacteria. In particular, phage KPO1K2 was found to give rise to a substantial halo surrounding plaques , as is sS. aureus biofilm, observed approximately 1-log greater biofilm reduction in terms of CFUs with phage SAP-26 presence than without. This difference was seen after 8 h of treatment, but not after 4 h, and continued through 12 and 24 h. As the phage titer appeared to be in excess of 108 PFU/mL (and I speculate was in fact 109 PFU/mL as 108 PFU seem to have been suspended in 100 μL), these results are indicative of phage anti-biofilm activity, and possibly phage bactericidal activity, but not necessarily also phage bacteriolytic or virion production activity.Rahman et al. , using 1Greater killing with phages being present than without phages being present, in both cases in the presence of rifampin, was also seen after 24 h of treatment as determined via a 2, 3, 5-triphenyltetrazolium chloride-based assay. Here, however, differences were only about two-fold, i.e., ~72%, ~60%, and ~35% cells living given phage-only, rifampin-only, or phage-plus-rifampin treatments. For azithromycin, these numbers instead are ~72%, ~75%, and ~40%, also suggesting phage activity compatibility with the presence of antibiotic. With vancomycin the numbers instead are ~72%, ~83%, and ~60%, which is less convincing of such compatibility of phage infection activity with antibiotic presence.E. coli biofilm bacterial numbers by about 2.5 log relative to an untreated control. In the presence of both phage T4 and the antibiotic, however, an approximately 6.5-log reduction in biofilm bacteria numbers was observed. This result should be indicative of phage population growth since the starting phage MOI was 0.01, which on its own at best should result in a killing of about 1% of the bacteria present (P. aeruginosa and phage PB-1 (and 0.5 μg/mL tobramycin), by contrast, yielded somewhat weaker evidence of phage activity in the presence of tobramycin. Coulter et al.  found th present . An equiS. aureus). Phage MR-5 and linezolid were applied to orthopedic-grade stainless steel Kirschner wires, with colony counts of adhered bacteria made after 6-, 24-, and 48-hs of incubation in the presence of bacteria. Both phage and antibiotic were applied to wires within hydroxypropyl methylcellulose hydrogels. Linezolid alone (5% w/w) and phages alone (1.5 × 109 PFU/mL) allowed similar levels of bacterial colonization of wires. A combination of the two agents, however, resulted in about 1-log less colonization . Bacteria which failed to encounter or be affected by linezolid upon attempting to adhere to wire surfaces thus seemingly could instead have encountered a phage, resulting in the additional reduction in CFUs.Kaur et al.  is a preThe implication of this result is retention of phage bactericidal activity in the presence of the antibiotic. Presumably, however, for phages to contribute to reducing bacterial colonization of wires, then it would have been those bacteria which were least impacted by linezolid that would have been most affected by phages, else those bacteria should have been sufficiently impacted by linezolid that phage infection would not have resulted in excess bacterial losses . That interpretation would call into question whether phage activity truly was retained given individual bacteria exposure to at least an MIC of antibiotic, with antibiotic concentration otherwise reported as “much higher than the MIC level”. It is also conceivable that phages were able to better gain access to bacteria and/or for subsequent bacteria killing to have occurred in the course of bacterial enumeration , i.e., aK. pneumoniae biofilm grown on microtiter plates with a combination of phage KPO1K2  and amikacin (40 μg/mL). Phages were either free or liposome entrapped upon application to biofilms, and biofilms were grown between one and seven days prior to the start treatment. The impact of antibiotic generally was less than that of phages, though not necessarily statistically significantly less, and free phages may have been slightly less effective as anti-biofilm agents than liposome-entrapped phages, depending on the age of the biofilm. That antibiotic treatment alone resulted in reductions in bacterial numbers found in biofilms, however, is suggestive that the dose of amikacin employed exceeded MIC, though MIC was not otherwise determined. Treated biofilms generally had bacterial counts that were closer to those of the negative-treatment controls, the older the treated biofilm, ranging from over 3-log reductions for one-day-old biofilms to roughly 1-log reductions for seven-day-old biofilms .Singla et al.  treated Experiments by Singla et al.  consistiWhy this possible phage virion production should have occurred in the presence of antibiotic especially in the case where phages were liposome entrapped, but not without liposome entrapment, is uncertain. This is especially uncertain given that, when phages were employed without antibiotic present, reductions in bacterial numbers ranged, as noted, from 1- to 3-log . Therefore, these experiments appear to be consistent with phage bactericidal and possibly also virion production activity in the presence of amikacin (though perhaps less virion production than as seen without antibiotic), but the result is complicated by its dependence on phage entrapment within liposomes. I speculate that the improved pharmacodynamic impact with phage liposome entrapment in the presence of amikacin, however, is a consequence of better phage penetration to bacteria. That is, either for spatial reasons (as consistent with authors’ suggestion) or for temporal reasons—as in declines in active antibiotic over time in combination with delayed phage adsorption to bacteria when liposome entrapped—bacteria were less affected by antibiotic at the point of phage encounter given phage liposome entrapment. A further complication is that phage KPO1K2 may encode an anti-biofilm-matrix hydrolase  see Sec.P. aeruginosa biofilm in the presence of various antibiotics, with phages NP1 and NP3 applied in combination to 106 PFU/mL. Specifically, they found that while ciprofloxacin and tobramycin blocked phage production at 8× MIC for these antibiotics, that blockage of phage production was not also seen at 1× MIC. Furthermore, for ceftazidime and colistin, phage production was seen even at 8× MIC, though with gentamycin no phage production was seen even at 1× MIC. This evidence, however, is not fully supportive of the idea that phage infections necessarily will be active in the presence of these antibiotics even at 1× MIC, given the potential for biofilm heterogeneity and antibiotic tolerance . In addition, for all five antibiotics at 1× MIC concentrations, phages were able to increase in number roughly 2- to 3-log fold over a 12-hour period following exposure to eight-hour biofilms growing on a layer of epithelial cells.Contrasting that conclusion, for ceftazidime and colistin 4-log increases in phage numbers were observed in the presence of biofilm and 8× MIC antibiotic concentrations, which suggests an involvement of all of the bacteria present in the observed phage population growth, i.e., as this was similar to the amount of phage population growth seen without antibiotic presence in a different experiment. This result therefore is highly suggestive of at least some phage population growth in association with fully antibiotic-exposed bacteria. As both antibiotics are externally rather than internally acting, however, this result may not be entirely unexpected, as consistent with the results of David et al. , as discClearly phage antibacterial activity or even production of new phage particles does not appear to be blocked absolutely under all circumstances of antibiotic co-treatment, as tested in this study. Nevertheless, for at least some of the experiments presented, questions remain as to the degree that antibiotics were affecting those individual bacteria responsible for supporting phage virion production.S. aureus biofilm with a combination of vancomycin and phage SATA-8505 resulted in roughly a half-log greater reduction in numbers of bacteria versus vancomycin alone. This was seen at vancomycin concentrations of 32, 64, and 128 μg/mL, while less reduction was seen at 256, 512, and 1024 μg/ml (MIC for non-biofilm bacteria was 4 μg/mL). Given that dosing was with only 106 PFU/ml, these results are suggestive of a retention of not just phage bactericidal activity but also phage virion production despite vancomycin presence. Similar results, however, were absent in the presence of equivalent quantities of cefazolin, dicloxacillin, linezolid, or tetracycline. As vancomycin is a cell-wall-targeting antibiotic, it is conceivable that suggested phage population growth occurred in these experiments prior to substantial vancomycin impact on bacterial metabolism.Kumaran et al.  found thP. mirabilis biofilm with phage vB_PmiS-TH (MOI = 1 or MOI = 100) and ampicillin (1× or 15× MIC). With MOI = 100, either with or without 1× MIC ampicillin, where 1× MIC ampicillin alone had little impact on biofilm bacteria, there was about 90% removal of biofilm after eight hours, as determined via crystal violet staining, but only 70% removal after 24 h. By contrast, with MOI = 100 and 15× MIC, there was no difference between eight and 24 h given this combined treatment, with 90% removal in both cases. Therefore, with time, and without substantial inhibition of bacterial growth as accomplished via inclusion of 15× MIC ampicillin, there appeared to be grow back of what we can presume are phage-resistant bacteria, i.e., 90% biofilm removal by eight hours, then only 70% biofilm removal by 24 h with MOI = 100 and antibiotic concentrations of either 1× or 0× MIC  versus MOI = 100 and antibiotic concentration of 15× MIC (“MOI 100 + AMP 246 μg/mL”) for which no grow back was observed. In the same experiment, with 15× MIC ampicillin, there was somewhat more biofilm removal after eight hours with MOI = 1 (50%) or MOI = 100 (90%) relative to ampicillin alone (20%). The latter results too are suggestive of phage bactericidal activity in the presence of antibiotic. Yazdi et al.  treated P. aeruginosa biofilms—saw greater reductions in biofilm biomass or biofilm viability with phage treatment  in combination with 1× MIC ciprofloxacin versus phage-only treatments, the latter which generally had little impact. These results would seem to imply that phages or antibiotic are unable to remove bacteria alone, and presumably thereby phages were unable to display bactericidal or bacteriolytic activity except when the other was also present. An alternative explanation is that with combination therapy then resistant bacteria are unable to reestablish biofilms following biofilm removal due to a low potential for bacteria to mutate simultaneously to both phage and antibiotic resistance . The same caveats about what these increases in phage numbers mean are applicable to these results as to those presented by Chaudhry et al. [Dickey and Perrot  tested iter 48 h  can be cy et al. , etc., t3 PFU/mL along with 1× MIC ciprofloxacin (0.5 μg/mL) and characterized the ability of simultaneously supplied P. aeruginosa to form biofilms (as then measured after 24 h of treatment). Approximately half as much biofilm biomass was present for all five tested phages  in the presence of ciprofloxacin in comparison with ciprofloxacin alone. No phage-only controls are presented with the shown experiment, but previous experiments indicated that phage-only treatment is less effective than ciprofloxacin treatment alone. Given the low numbers of phages provided, these results are suggestive that phage virion production indeed may have occurred despite ciprofloxacin’s presence. That the bacteria formed biofilms at all in the presence of ciprofloxacin, however, is puzzling, perhaps suggesting that it was instead ciprofloxacin-resistant mutants which formed the biofilm, and also ciprofloxacin-resistant mutants which supported the proposed phage population growth.Issa et al.  applied The results reviewed in this subsection are highly suggestive that phage infection activity can persist in association with bacterial biofilms despite presence of antibiotic concentrations of 1× MIC or greater. Nevertheless, issues exist concerning the heterogeneity of biofilms and cultures, both spatially and temporally. These are in terms of the degree that constituent bacteria are potentially unevenly impacted by antibiotic while supporting phage infection activity, as well as the potential for antibiotics to decay such as to subinhibitory levels over time. In either case, phage infection activity might be occurring in association with antibiotic not inhibitorily affecting bacterial hosts despite initial antibiotic concentrations at or exceeding 1× MIC. Additional issues are the presence of biofilm-matrix degrading enzymes in at least some phage formulations as may mimic phage bactericidal activity, and also the potential for antibiotic-resistant bacteria to sufficiently grow in number over time to support phage virion production in an antibiotic-unaffected manner. Ideally, therefore, determinations of antibiotic impact on phage infection activity would be corroborated at the very least with simplified, shorter term determinations of phage-antibiotic compatibility such as the above-mentioned single-step growth experiments ,91.For phage therapy to be effective, then phages at a minimum need to reach targeted bacteria and then kill those bacteria. Generally, the killing process involves several relatively complex physiological steps that are highly dependent on various target-bacterium-supplied functions, especially as resulting in phage gene expression. In addition, to some degree phage in situ replication can be helpful or even crucial to phage therapy success, resulting in so-called active treatments ,94,95,96K. pneumoniae, lobar-pneumonia model, found no difference in phage SS impact on bacteria with versus without antibiotic presence. A reported 1× MIC amikacin (3.75 mg/25 g) was supplied via intraperitoneal delivery at the same time as phages (109 PFU), also intraperitoneally, both in association with a simultaneous intranasal bacterial challenge (5 × 106 CFU). Bacterial counts on Day 5 with phage treatment were 5-logs, or more, lower relative to the no-treatment control. Clearly phage bactericidal activity therefore was retained in this model in association with antibiotic. Antibiotic alone, however, had little impact on bacterial numbers. Evidence also is provided that, in the absence of bacteria, phages could concentrate in lung tissue, up to nearly 1012 PFU/g. As a result, it is uncertain whether phage replication may have occurred in the presence of antibiotic to result in observed declines in bacterial presence versus phage concentration to inundative titers in the lungs by other means, or, if phage replication did occur, whether antibiotic concentrations in contact with phage-infected bacteria were still equal to at least 1× MIC at the time of that replication. In addition, measured reductions in bacterial loads relative to untreated controls—given phage-only or combined treatments—did not begin until Day 2 of treatment, and reductions were not substantial, relative to bacterial numbers as present on Day 1, until Day 3 of treatment. Over this time frame, antibiotic concentrations likely would have declined to below 1× MIC, as evidenced by bacterial growth in association with the amikacin-only treatment group by Day 2. Therefore, evidence that phage replication occurred in association with 1× MIC or greater concentrations of amikacin in this study at best is not robust.Chhibber et al. , using aS. aureus model diabetic foot infection, mouse model, using a combination of phage MR-10  and linezolid , with treatment starting 30-min post bacterial challenge. Evidence of phage virion production in the presence of antibiotic at best is slight as determined by phage titers found locally, as too is evidence of phage replication in terms of reductions in CFUs, also as found locally, where reductions were not substantial relative to likely starting bacterial densities. Combination treatments reduced bacterial densities by about 1-log more than phage- or antibiotic-alone treatments, however. This latter result, particularly as it occurred within one day of the bacterial challenge, and at the start of treatments, is suggestive of phage bactericidal activity in the presence of both antibiotic and reasonably antibiotic-affected bacteria as found in vivo.Chhibber et al.  treated S. aureus and phage Sb-1) but less robust results against a second (P. aeruginosa and phage vB_PsaP PAT14). Phages were applied directly to infections by injection, once per day for three days (107 PFU/dose), and antibiotics were supplied intraperitoneally once per day for 14 days . With S. aureus, numbers were reduced relative to the untreated control by 7 logs given antibiotic-only treatment and by 9 logs with combination treatment . With P. aeruginosa, by contrast, 3 log reductions were seen with antibiotic-only treatment, but only 4 log reductions with either phage-only or combination treatments. These results might be consistent with phage virion production in vivo, though without volume or in situ phage titer information this possibility is not necessarily easily corroborated. The results nevertheless should be viewed as consistent with at least phage bactericidal activity particularly against S. aureus.Yilmaz et al. , in sepaS. aureus surface adherence to orthopedic-grade stainless steel Kirschner wires, i.e., as discussed above in terms of in vitro testing [w/w of wires), which in turn was slightly more effective than impregnation with phage MR-5 alone (109 PFU/mL). Perhaps consistently, a faster drop off in phage titers in vivo was seen with antibiotic present versus without, but still with increases in titers in both cases early on during experiments. The in vivo results overall are suggestive of slightly lower phage activity in the absence of linezolid in comparison to what was observed in vitro [Kaur et al.  presents testing  (Section-5 alone  PFU/mL. in vitro  and [109in vitro  providesP. aeruginosa rat endocarditis model. After six hours of treatment, started 18 h post bacterial challenge, bacterial loads were reduced by 2–3 logs by either phage or antibiotic treatment, but by roughly 7 logs by combined phage and antibiotic treatment. Phage titers in plasma were approximately 108 PFU/mL, but ~1010 PFU/g in association with aortic valve vegetations versus ~109 CFU/g. Based on Poisson distributions (Oechslin et al.  exploredibutions , it is d7-fold reduction in bacterial numbers is in excess of 103 × 103 = 106 approximate reduction as predicted for additive phage-antibiotic interactions.Ciprofloxacin was dosed at 20 mg/kg with a single bolus at the same time as phages were applied and calculated MIC was 0.19 μg/mL. Given the short duration of the experiment as well as the substantial impact that antibiotic alone had on bacterial numbers, it seems reasonable to conclude that this proposed aortic valve vegetation-located phage replication occurred in association with bacteria that at least could have been exposed to 1× MIC or greater ciprofloxacin, as would be consistent with their in vitro, broth results presented in the same study . These rE. coli mouse gastrointestinal colonization, neither support nor refute compatibility of phage activity with presence of ciprofloxacin. The phage used was not named and treatment doses were delivered orally by gavage.The results of Vahedi et al. , though Animal experiments, as noted, are highly complex in terms of the state of the bacteria interacting with treatment phages. Therefore, the potential for phages to display infection activity while infecting bacteria that are not substantially affected by antibiotic presence, despite those bacteria being antibiotic susceptible, could be high. That results of these in vivo experiments are supportive of phage infection activity during treatments, and even in certain cases virion production, therefore might not be surprising. It is notable, however, that the more impressive of the animal co-treatment results  is assocIt is unfortunate that much of the strongest evidence of phage infection activity in the presence of antibiotics comes from the most complex experimental systems: in vitro biofilms or in vivo disease models. On the one hand, the results obtained suggest that there can be substantial latitude in terms of the potential for phages to kill bacteria and/or to produce new virions despite antibiotic presence. In particular, perhaps phages are infecting especially those bacteria which are least affected by antibiotic treatments. On the other hand, the ambiguities associated with the interpretation of many studies are highly suggestive of a utility to better characterizing phage compatibility with antibiotic treatments under more rigorous conditions, e.g., as via single-step growth experiments ,91 as doSummers , in 2001The utility of phage-antibiotic co-treatments thus is being increasingly explored in the pre-clinical phage therapy literature ,41. SubsAlthough phage virion production during phage therapy exists as an ideal, it nevertheless is possible for phages to kill bacteria without necessarily also producing phage progeny, resulting in what are known as abortive infections ,43. A coPhages may primarily be used to target those bacterial subpopulations, in situ, which for whatever reason—e.g., poor vascularization, biofilm residence, decay in antibiotic densities over time—are exposed to lower concentrations of antibiotic ,59. In t


The prevalence of bacterial etiology results in a large consumption of broad-spectrum antibiotics, which in turn leads to increased rates of resistant uropathogens. Therefore, non-antibiotic prevention and treatment options are now of great importance.Urinary tract infections (UTIs) are one of the more common infections encountered in everyday clinical practice. They account for 10–20% of all infections treated in primary care units and 30–40% of those treated in hospitals. The risk of UTI in the female population is considered to be 14 times higher than in the male populationA systematic literature search was performed for the last 20 years (1999–2019) and the efficiencies of these eight different non-antibiotic interventions were analysed and discussed.d-mannose, estrogens, vitamins, and immunotherapy.This article provides an overview on non-antibiotic options for management of UTI, including the application of cranberry products, the phytodrug Canephron N, probiotics, nonsteroidal anti-inflammatory drugs (NSAID), The last 20 years of research on non-antibiotic approaches in UTI have not brought conclusive evidence that antibiotic usage can be replaced completely by non-antibiotic options. Hence, antibiotics still remain a gold standard for UTI treatment and prevention. However, changing the therapeutic strategy by including non-antibiotic measures in the management of UTI could be successful in avoiding antimicrobial resistance at least to some extent. In everyday clinical practice, urinary tract infections (UTIs) are seen very frequently. They account for 10–20% of all infections treated in primary care units and 30–40% of those treated in hospitals  [The main substances responsible for the inhibition of adhesion of f A-type . Vostalo= 0.01)] .The overall results suggest that cranberry products may be an option for prevention of UTI in healthy, non-pregnant patients, as well as in patients after gynecological surgery when a catheter was placed. However, these findings still need confirmation because the conducted studies did not involve a large enough number of participants. What is more, Liska et al. in their meta-analysis on cranberries and UTIs stated that a number of publications may report conflicting conclusion due to the fact that recommendations are mainly directed to women with rUTI and that they include outcome from various populations which also can cause differences in the results .Another alternative, non-antibiotic approach to UTI treatment is the administration of herbal preparations. One product approved in many countries is Canephron N . This contains century herbs, lovage roots and rosemary leaves. It has diuretic, spasmolytic, anti-inflammatory, antibacterial and nephroprotective properties, and is also considered to be safe in both pregnancy and during breastfeeding .Miotla et al. evaluated the efficiency of Canephron N in the prevention of UTI in high-risk female patients undergoing urodynamic studies (UDS). Therein, women with at least one risk factor for UTI received after UDS either 3 g of fosfomycin trometamol (FT) single dose or 5 ml of Canephron N taken orally three times daily for 1 week. There was no statistically significant difference in UTI incidence between the two groups. The authors concluded that prophylaxis of UTI after UDS with this phytodrug may be a good alternative to antibiotics administered after UDS in high-risk female patients .Wagenlehner et al. conducted a double-blinded, multicenter, non-inferiority study comparing the efficiency of UTI treatment with Canephron N to standard antibiotic therapy with fosfomycin trometamol. A large cohort of female patients with symptoms of acute uncomplicated lower UTI was randomly allocated into two groups receiving either Canephron N 2 dragees t.i.d. or fosfomycin trometamol 3 g single dose with corresponding matched placebo. The results were very promising, as only 16.5% of patients treated with Canephron N required additional antibiotic treatment as compared to 10.2% in the fosfomycin group. The study, hence, demonstrated non-inferiority  of the herbal compared to the antibiotic treatment. Of note, an additional advantage was that with Canephron N, fewer gastrointestinal side effects, such as diarrhoea and abdominal pain, were observed. In the phytodrug group, however, five episodes of pyelonephritis occurred (mainly during the first days of therapy), as compared to one episode of pyelonephritis in the fosfomycin group .Lactobacillus spp. colonies are associated with an increased risk of UTI. These changes can be induced by antimicrobial therapy, hormone level changes due to menopause or usage of contraceptives and even by UTI itself [The main argument in favour of using probiotics for UTI prevention and treatment is the fact that the vagina is a potential reservoir for the bacteria bringing about UTIs. Changes in the vaginal microbiota with reduction of protective I itself . The use9 colony-forming units of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. The results of the study showed that supplementation with 480 mg L. rhamnosus GR-1 and L. reuteri RC-14 significantly decreased the mean number of recurrences in patients with uncomplicated UTIs when compared with trimethoprim-sulfamethoxazole administration. However, unlike trimethoprim-sulfamethoxazole, lactobacilli did not increase antibiotic resistance. In the study, after 1 month of trimethoprim-sulfamethoxazole prophylaxis, resistance to trimethoprim-sulfamethoxazole, trimethoprim and amoxicillin increased from approximately 20–40%, to approximately 80–95% in E. coli from the feces and urine of asymptomatic women and those with E. coli-induced UTIs [In a double-blind, non-inferiority trial conducted by Beerepoot et al. 252 postmenopausal women with rUTIs were randomized to receive 12 months of either antibiotic prophylaxis with trimethoprim-sulfamethoxazole or oral probiotic capsules containing 10ced UTIs .Lactobacillus crispatus strains) or placebo for 5 days, then once weekly for 10 weeks. Results of this study showed a significant reduction of UTI episodes in patients who received intravaginal Lactobacillus treatment, compared to the placebo group. Herein, Lactin-V treatment resulted in prolonged colonization with L. crispatus strains and it reduced the frequency of rUTI by around 50% among participants. Unfortunately the results could not be compared directly with antibiotic prophylaxis of UTIs [In another double-blinded study, Stapleton et al. investigated premenopausal women with a history of rUTIs, by giving them daily either Lactin-V  with diclofenac  for 3 days. Here, fosfomycin trometamol was used as rescue antibiotic (3 g single dose) after completion of the study drug on day 3, if symptoms persisted. The study showed that diclofenac was inferior to norfloxacin in terms of symptoms relief, but it could reduce the usage of antibiotics, although it increased the risk of pyelonephritis (6 cases in the diclofenac and none in the norfloxacin group) [In a double-blind, multicentre trial by Gagyor et al. female patients with UTI symptoms were randomized into groups receiving either fosfomycin trometamol  or ibuprofen . Only patients with no risk and complicating factors were recruited. Herein, two-thirds of the ibuprofen group recovered without antibiotics, but had a significantly higher total burden of symptoms, while more had pyelonephritis (five cases in the ibuprofen and one in the fosfomycin group). The authors suggest that symptomatic treatment is a possible option to be considered in women with mild to moderate UTI symptoms . Bleidorn group) .Replacing antibiotics with NSAIDs for the treatment of uncomplicated UTI may be at the cost of prolongation of symptoms and increased risk of pyelonephritis. Due to that fact, in uncomplicated UTI it is preferable to delay the use of antibiotic while closely monitoring the patient rather than completely resigning from antimicrobial treatment. In this strategy it is also important to share a decision-making process with a patient and to know his/her expectations towards the treatment . Of noted-Mannose is a monosaccharide that can be rapidly absorbed and excreted by the urinary tract and can prevent the adhesion of type 1 bacterial fimbria to the uroepithelium. This is a bacterial virulence factor promoting UTI—especially caused by E. coli [The urinary tract mucus membrane is coated with proteins that interfere with the adhesion of bacteria . d-Manno E. coli .d-mannose daily (n = 103), the second group got 50 mg of nitrofurantoin daily (n = 103) and the third group did not receive any prevention (n = 102) for 6 months. The results of the study showed that the risk of UTI was significantly reduced by both d-mannose and nitrofurantoin, with a lower risk of side effects in the d-mannose group. The authors conclude that d-mannose may be useful for UTI prevention, although more research is still required [Kranjcec et al. randomly allocated female participants with rUTIs to three groups. The first group received 2 g of powdered required .Enterobacteriaceae colonization [Estrogen is a hormone not only responsible for regulating the female reproductive system, but also for stimulating the proliferation of lactobacilli, reducing vaginal pH and decreasing vaginal nization . Estrogenization  StimulatCurrent literature shows both the reduction of UTI incidence after topical intravaginal estrogen use , 39 and Vitamin C (ascorbic acid) supplementation is often recognized as a non-antibiotic prophylaxis for rUTIs. It has two suggested mechanisms of action. The first is urine acidification  and the Vitamin D is also recommended as a supplement for rUTI prevention. This is based on its function as an inducer of antibacterial innate immune responses . Jorde eBacterial extracts are used in the management of UTI because they are able to stimulate the host’s immune system through the activation of monocyte-derived dendritic cells to produce antibodies and cytokines . They maE. coli (UPEC) strains [In their review and meta-analysis, Naber et al. identified 11 studies which looked into the use of both oral and vaginal bacterial lysates in the prophylaxis of rUTIs . Three o strains . Five ou strains –56. Acco strains .Aziminia et al. also systematically reviewed the evidence regarding the efficacy of vaccines or immunostimulants in reducing the recurrence rate of UTIs. They included five studies using OM-89. In four of these studies, a significant reduction of rUTIs was demonstrated –56. In oE. coli, Proteus vulgaris, Klebsiella pneumoniae, Morganella morganii, and Enterococcus faecalis, three randomised, clinical trials were analysed. Therein, one study (n = 91) used high-dose vaccine, low-dose vaccine and placebo [n = 54 and n = 75) used vaccine with booster, without booster and placebo [Of another vaccine preparation, Urovac, consisting of ten heat-killed uropathogenic species—including six serotypes of  placebo ; whereas placebo , 60. OveE. coli O-antigen in the form of an intramuscular biconjugate vaccine containing O-antigens of four E. coli serotypes (ExPEC4V). The vaccine was well tolerated and there were no serious adverse effects in the experimental group. It also gave a functional antibody response that resulted in significantly fewer UTIs by different E. coli serotype in the vaccine group, compared with the placebo group. The evidence from this trial suggests that ExPEC4V did not reduce UTI recurrence compared to placebo at study endpoint , but the number of UTIs caused by different serotypes of E. coli was significantly lower in the vaccine group compared with the placebo group . Huttner et al. have now initiated phase II studies to confirm these promising findings [In contrast to the aforementioned results, Huttner et al. conducted a randomized, single-blind, placebo-controlled phase 1b trial on 188 healthy female patients with rUTIs. The aim was to assess the safety and immunogenicity of findings .Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Enterococcus faecalis. The mechanism of action of Uromune is based on a theory that stimulation of the sublingual mucosa will lead to an activation of an immune response in the urinary tract mucosa [Uromune is another bacterial extract with a potential benefit when used in UTI management. It is a sublingual spray composed of inactivated bacteria—t mucosa . In Yangt mucosa .® and SolcoUrovac® containing ten heat-killed uropathogenic bacteria, Urostim® containing four microbial species, and Urvakol® containing 5 Gram-negative species and for adjuvant activity Propionibacterium acnes, no trials complying with the predefined criteria could be found within this period of time.For other bacterial lysates recommended for prophylaxis of rUTI, such as StroVacUTIs are a common problem for women at different ages. Yet, UTIs are not only a problem unique for the patient, but are also a factor of high cost for the health care system. Increasing antimicrobial resistance with its expenditure and health consequences has raised interest in applying different non-antibiotic ways of preventing and treating uncomplicated lower UTIs. Unfortunately, the last 20 years of research on non-antibiotic approaches in UTI have not brought conclusive evidence that antibiotic usage can be replaced completely by non-antibiotic options. Hence, antibiotics still remain a gold standard for UTI treatment and prevention. However, changing the therapeutic strategy by including non-antibiotic measures in the management of UTI could be successful in avoiding antimicrobial resistance at least to some extent. According to the 2019 updated guidelines of the European Association of Urology, the prevention of rUTI includes first of all counseling of risk factors avoidance, then non-antimicrobial measures and finally antimicrobial prophylaxis. These interventions should be incorporated with an emphasis on that order . With th


Antimicrobial stewardship programs potentially lead to appropriate antibiotic use, yet the optimal approach for neonates is uncertain. Such a program was implemented in a tertiary care neonatal intensive care unit in October 2012. We evaluated the impact of this program on antimicrobial use and its association with clinical outcomes.In a retrospective cohort study, we examined 1580 neonates who received antimicrobials in the 13-months before and 13-months during program implementation. Prospective audit and feedback was given 5 days a week on each patient who was receiving antibiotic. Pharmacy and microbiology data were linked to clinical data from the local Canadian Neonatal Network database. The primary outcome was days of antibiotic therapy per 1000 patient-days; secondary outcomes included mortality, necrotizing enterocolitis, and antibiotic duration for culture-positive and culture-negative late-onset sepsis. The breadth of antibiotic exposure was compared using the Antibiotic Spectrum Index.P < 0.001), without an increase in mortality. There was no difference in duration of therapy in culture-negative or culture-positive sepsis, rates of necrotizing enterocolitis, or breadth of antibiotic exposure. Fewer antibiotic starts occurred during program implementation . The use of narrow-spectrum agents decreased (P < 0.001) whereas the use of cefotaxime increased (P = 0.016) during program implementation.Overall antibiotic use decreased to 339 days of therapy per 1000 patient-days from 395 (14%, Daily prospective audit and feedback was not associated with a change in antibiotic duration or clinical outcomes, however there were fewer babies started on antibiotics, suggesting that additional interventions are required to inform and sustain changes in antibiotic prescribing practices. In North American hospitals, antimicrobial stewardship programs (ASPs) have become a required practice for accreditation . While aUp to 72% of neonates admitted to intensive care are prescribed antibiotics  and up tEvaluate the impact of the ASP on antimicrobial use in days of therapy per 1000 patient-days, and its association with clinical outcomes, including late-onset neonatal sepsis (LONS), candidemia, NEC and death; and.determine the impact of an ASP on the duration of therapy for culture-positive and culture-negative sepsis.In 2009, a multidisciplinary ASP was established at Mount Sinai Hospital in Toronto, Canada; it was expanded through a broader provincial initiative into the NICU in October 2012 . Using pThe study was approved by the hospital’s Research Ethics Board.Our study included neonates admitted to the 55-bed inborn Level II and III NICU at Mount Sinai Hospital, started on an antibiotic and followed until discharge from the NICU. Approximately 1100 newborns are admitted annually to the unit, which cares for non-surgical preterm and sick term neonates. In 2011, prior to introducing the ASP, the rate of mortality was 5.4%; NEC was 1.9%, culture-positive early- and late-onset sepsis were 0.8 and 7.2%, respectively. Neonates with surgical conditions are sent to a tertiary pediatric hospital. Babies who did not receive an antibiotic were excluded from the study.The pre-intervention period covered September 1, 2011, to October 21, 2012, and October 22, 2012 to November 30, 2013 reflected the ASP implementation period. Empiric therapy for early-onset neonatal sepsis  was ampicillin and gentamicin; empiric therapy for LONS (more than 72 h of age) was cloxacillin and gentamicin, or vancomycin and gentamicin if a central catheter was present. All neonates received one blood culture when evaluated for sepsis; a second blood culture was drawn to confirm clearance if the initial blood culture was positive. Cefotaxime was added empirically if there was concern of meningitis from the clinical presentation or cerebrospinal fluid analysis.Starting in October 2012, a pediatric infectious diseases-trained physician and/or a pharmacist specializing in antimicrobial stewardship performed PAF fives day a week on every neonate who received antibiotics. They participated in dedicated ASP rounds five mornings a week with the NICU medical team, which included staff physicians, fellows, nurse practitioners, residents and students. Neonates who were being considered for antibiotic treatment were not reviewed. The discussions included reviews of the evidence with the team, such as use of serial C-reactive protein for suspected sepsis, or clarithromycin to prevent bronchopulmonary dysplasia; discussions around microbiology and pathophysiology, such as the management of a baby born to a mother with chorioamnionitis with in-utero antibiotic exposure and negative neonatal blood cultures; support of changes in practice by individuals, such as discontinuing antibiotics with negative cultures in the context of clinical sepsis; and affirmed these changes in presentations at staff meetings. The antibiotic courses for babies followed by the infectious diseases service were not reviewed but were included in the analysis. Suggestions were made around antimicrobial use, including, but not limited to, choice of antimicrobial and duration of therapy. Eight months into the program, the frequency of ASP rounds was decreased to 4 days a week.Antibiotic use was determined using days of therapy (DOT) and antibiotic days. DOT was calculated as the number of days the neonate was on a specific agent. For example, ampicillin for 3 days resulted in 3 DOT, irrespective of number of doses received in one day. An antibiotic day is defined as exposure to antimicrobials on a specific day  and does not take into consideration the number of antimicrobials. Thus, a neonate who received ampicillin and gentamicin for 3 days would have 3 antibiotic days but 6 DOT.Episodes were based on a count of consecutive dispense dates at the patient level and defined as suspected sepsis if antibiotic days were less than or equal to 3 to reflect empiric therapy pending culture results. If antibiotics were continued for 4 or more days, this was counted as an episode of culture-positive or culture-negative sepsis, depending on the laboratory result that accompanied the initial dispense date. If there were 3 or more days between dispense dates, then it was assumed to be a new episode.Antimicrobial data were obtained from the Mount Sinai Hospital pharmacy database  for each patient, and included the name of the agent, the route of administration and the date(s) dispensed. Topical antibiotics, prophylactic antibiotics, immunizations, antifungals, antivirals and anti-retrovirals were excluded from the analyses. Patient-level pharmacy data were linked to microbiological data from the laboratory information system . Data on all positive cultures from sterile sites during the neonate’s admission were collected. These were also linked to clinical data extracted from the Canadian Neonatal Network (CNN) database, which uses standardized definitions to capture information from neonate medical records . Study vTo assure data quality and adequacy, the data abstractor adhered to definitions from the CNN’s Abstractor’s Manual for clinical characteristics and outcomes of interest . The datOur primary outcome measure was antibiotic DOT per 1000 patient-days (the sum of the DOT accounting for each antibiotic over 1000 patient-days). Secondary outcomes were proportion of neonates who received antibiotics during their admission; number of antibiotic days and total length of stay; mortality prior to discharge; NEC stage 2 or 3; duration of therapy for LONS, defined as an antibiotic course of 3 or more days in a symptomatic neonate aged 3 days or greater; and the incidence of antibiotic restarts within 3 days after discontinuation, as a balancing measure. We also examined duration of therapy — i.e. total consecutive antibiotic days — for culture-positive and culture-negative sepsis episodes.We evaluated the breadth of antibiotic exposure using the Antibiotic Spectrum Index (ASI), which classifies antibiotics according to spectrum of activity, and has been shown to be sensitive to antibiotic selection for specific conditions, as compared to DOT per 1000 patient-days . Using tClinical characteristics were summarized using descriptive statistics; the Mann-Whitney U test and Pearson’s chi-squared test or Fisher’s exact test was used, as appropriate, for significance testing between the two periods.p-values less than 0.05 were considered statistically significant. All statistical analyses were performed using R statistical software version 3.4.2  [Antibiotic DOT per 1000 patient-days pre- versus during ASP was analyzed using a Poisson model, and 95% confidence intervals were reported. This was done for the overall comparison and subgroups. Two-sided Austria) .Birthweight, gestational age and SNAP II scores on admission were similar between the pre- and during ASP groups. The length of stay and number of antibiotic days between the groups were similar pre-ASP and during the implementation period Table . Rates oP < 0.001), particularly in the 29–33 weeks gestational age group.There were 2582 neonates admitted to the NICU during the study period; 1580 (61%) were started on antibiotics at some time during their stay (864 pre-ASP and 716 post-ASP). Following ASP implementation, antibiotic consumption decreased by 56 DOT per 1000 patient-days (14%) among all gestational age groups, most significantly among extreme premature and near-term neonates (Table p < 0.001), and an increase in the use of cefotaxime (p = 0.016) and non-significant increase in the use of vancomycin (p = 0.353) during ASP implementation.Gentamicin, ampicillin, cefotaxime and vancomycin were the most frequently prescribed antibiotics, reflecting 87 and 90% of antibiotics in the pre- and during ASP implementation periods, respectively Fig. . There wStreptococcus agalactiae, one with Escherichia coli) within 4 to 6 days of discontinuing antibiotics. Bacteremia occurred more frequently in the pre-ASP period, although not statistically significant  in the context of culture-positive bacteremia and culture-negative LONS, and identified no difference in the burden of antibiotic exposure following ASP implementation for both clinical conditions. This metric illustrated a significant decrease in the spectrum of antibiotic prescribing patterns for pediatric community-associated pneumonia as compared to DOT per 1000 patient-days, following an ASP intervention that led to a change in prescribing practice from ceftriaxone (ASI = 5) to ampicillin (ASI = 2) . The ASIThe strengths of this study lie in its multi-disciplinary involvement with an infectious diseases physician and ASP pharmacist, and incorporation of PAF into the morning clinical rounds, which provided an opportunity for active engagement of neonatal staff and trainees in the discussions. Objective clinical measures were used, as well as a standardized method of reporting antibiotic consumption. Despite being a single institution study, the setting reflects many other Level III NICUs, with a broad mix of inborn non-surgical patients of variable gestational ages.A number of limitations relate to measurement tools and the study intervention. Dispense data were acquired at the patient level, which may not have been correlated with patient administration; however, we have no reason to believe that this changed between the study periods. We did not document the content of our advice, whether recommendations were agreed upon, and associated clinical outcomes. Review of this information may have helped the ASP to target specific prescribing behaviors using other modalities in addition to PAF. There was also variability in the nature of the PAF discussions based on the readiness of each staff neonatologist to change his/her prescribing practices, which may have affected the duration of therapy and antibiotic choice at various time points.The clinical context surrounding antibiotic courses was not available for analysis. For example, cerebrospinal fluid studies were not included, so a patient may have appropriately received cefotaxime in the context of suspected meningitis and analyzed as “culture-negative sepsis”, alongside other cases of inappropriately prolonged cefotaxime therapy. Such information would help to determine if the increase in cephalosporin use during the ASP implementation period was appropriate for the clinical context.The NICU ASP, with daily prospective audit and feedback by a multidisciplinary team, was associated with a significant decrease in antibiotic consumption despite a lack of significant change in antibiotic duration of culture-negative sepsis and an increase in cefotaxime use. Our findings suggest that PAF alone cannot inform antibiotic prescribing behaviours around discontinuation of antibiotics in neonates. There is a need to define and measure appropriateness of antibiotic use that incorporates clinical context, diagnostic tools, antibiotic choice and duration of therapy into real-time audit and feedback. Such a metric should be generalizable, able to discriminate the risk of bacterial sepsis from other non-infectious complications, particularly in preterm infants, and inform strategies to reduce antibiotic misuse in critically ill neonates.