One of the main limitations of phage therapy is the rapid emergence and proliferation of BIMs. Pires et al. (41) reported P. aeruginosa biofilm regrowth 6 h after application of a single phage treatment in vitro, which was attributed to the proliferation of BIMs lacking phage receptors. The same has been reported for phage-treated biofilms of K. pneumoniae, where a rapid regrowth was observed following the initial lysis, suggesting that phage-resistant variants were selected in the host populations (109).
噬菌体疗法的主要局限之一是生物膜的快速出现和增殖。Pires 等人（41）报道了铜绿假单胞菌生物膜在体外应用单一噬菌体处理 6 小时后重新生长的情况，这归因于缺乏噬菌体受体的 BIMs 的增殖。肺炎双球菌经噬菌体处理后的生物膜也出现了同样的情况，在最初的溶解后迅速再生，这表明宿主群体中选择了抗噬菌体的变种（109）。
The clinical significance of BIMs remains unclear. Several studies have shown that phage resistance may diminish fitness or virulence of these bacterial variants and therefore facilitate clearance by the immune system (110). Olszak et al. (111) demonstrated that a P. aeruginosa biofilm population that survived PA5oct jumbo phage treatment became sensitive to the immune system due to the reduced virulence of BIMs. Despite the fact that resistance can be associated with decreased bacterial virulence, phage resistance should not be underestimated and efforts should be made to develop methodologies for preventing it.
BIMs 的临床意义尚不清楚。一些研究表明，噬菌体抗性可能会降低这些细菌变种的适应性或毒力，从而促进免疫系统的清除 ( 110)。Olszak 等人（111）证实，由于 BIMs 的毒力降低，经 PA5oct 巨型噬菌体处理后存活下来的铜绿假单胞菌生物膜群体对免疫系统变得敏感。尽管抗药性可能与细菌毒力下降有关，但不应低估噬菌体抗药性，应努力开发预防噬菌体抗药性的方法。
The application of cocktails composed of phages that target different cell receptors has been suggested to improve phage therapy by extending host range and reducing resistance (112). This is particularly important in biofilms, in which the application of cocktails rather than a single phage can delay (41) or even prevent the emergence of bacteria-resistant variants. Morris et al. (113) evaluated the therapeutic effect of a phage cocktail for treating peri-prosthetic joint infections caused by S. aureus in rats and demonstrated that the bacterial isolates recovered from the infected knee of the animals that received phage therapy remained susceptible to the five-phage cocktail.
有人认为，应用由针对不同细胞受体的噬菌体组成的鸡尾酒可以扩大宿主范围并减少抗药性，从而改善噬菌体疗法（112）。这一点在生物膜中尤为重要，在生物膜中应用鸡尾酒噬菌体而非单一噬菌体可以延缓（41）甚至防止细菌耐药性变种的出现。Morris 等人（113）评估了鸡尾酒噬菌体治疗由大鼠金黄色葡萄球菌引起的假肢关节周围感染的疗效，结果表明，从接受噬菌体治疗的动物受感染膝关节中分离出的细菌对五种噬菌体鸡尾酒仍然敏感。
Another way to prevent phage resistance is combining phages with antibiotics. Verma et al. (114) prevented the emergence of phage-resistant variants during treatment of K. pneumoniae biofilms by combining ciprofloxacin with phages. The application of phages with antibiotics, simultaneously or sequentially, has been described as particularly effective against biofilms. Synergism may occur because phage-associated bacterial lysis releases nutrients that can reactivate the metabolic activity of the growth-arrested cells, which become sensitive to antibiotics. Cell lysis also causes a dispersion of the EPS, enhancing the diffusion of the antibiotic to the inner matrix layers, whereas the oxygen availability increases the drug activity (108). In some cases, phage-resistant cells might be more susceptible to antibiotics (115).
防止噬菌体抗药性的另一种方法是将噬菌体与抗生素结合使用。Verma 等人（114）通过将环丙沙星与噬菌体结合使用，防止了肺炎克氏菌生物膜治疗过程中噬菌体抗药性变体的出现。据描述，噬菌体与抗生素同时或先后使用对生物膜特别有效。之所以会产生协同作用，是因为噬菌体相关的细菌裂解释放出的营养物质可重新激活生长受阻细胞的代谢活动，使其对抗生素变得敏感。细胞裂解还会导致 EPS 分散，从而加强抗生素向基质内层的扩散，而氧气的供应则会增加药物的活性（108）。在某些情况下，抗噬菌体的细胞可能对抗生素更敏感（115）。
Besides resistance, the efficacy of phage therapy against biofilms can be compromised by the deficient phage penetration into the biofilm matrix, as already mentioned. Nevertheless, mechanical or enzymatic disruption of the biofilm can facilitate phage infection, which was already proven both in vitro and in vivo. Melo et al. (20) reported poor antibiofilm activity by a sepunavirus, despite its high activity against planktonic cells at different growth stages. It is noteworthy that the authors demonstrated that after mechanical disturbance, the biofilm becomes susceptible to phage attack. In a study by Seth et al. (116), the application of a phage treatment in S. aureus biofilm wounds had no effect on healing; however, when the phage was administered after sharp debridement, wound healing parameters assessed by histological analysis improved significantly and bacterial counts were reduced.
除了抗药性之外，噬菌体对生物膜的治疗效果也会因为噬菌体对生物膜基质的穿透力不足而受到影响。不过，机械或酶法破坏生物膜可以促进噬菌体感染，这在体外和体内都已得到证实。Melo 等人（20）报告称，尽管一种sepunavirus对不同生长阶段的浮游细胞具有很高的活性，但其抗生物膜活性很差。值得注意的是，作者证明了在机械干扰后，生物膜容易受到噬菌体的攻击。在 Seth 等人的一项研究（116）中，在金黄色葡萄球菌生物膜伤口中应用噬菌体治疗对伤口愈合没有影响；但是，如果在锐性清创后使用噬菌体，通过组织学分析评估的伤口愈合参数会明显改善，细菌数量也会减少。