Natural Antibiotic Resistance of Bacteria Isolated from Larvae of the Oil Fly, Helaeomyia petrolei

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 μg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene–50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.

Helaeomyia petrolei（油蝇，oil fly）幼虫栖息于美国加利福尼亚州洛杉矶兰乔拉布雷亚（Rancho La Brea）的沥青渗漏点。此前研究者对从粘稠沥青中采集的油蝇幼虫可培养肠道微生物群落进行了分析，14株分离菌株中的9株被鉴定为普罗威登斯菌属（Providencia spp.）。随后，研究者对12株分离得到的细菌菌株进行了23种常用抗生素的敏感性测试。结果显示，9株归类为雷氏普罗威登斯菌（Providencia rettgeri）的菌株对四环素（tetracycline）、万古霉素（vancomycin）、杆菌肽（bacitracin）、红霉素（erythromycin）、新生霉素（novobiocin）、多粘菌素（polymyxin）、粘菌素（colistin）以及呋喃妥因（nitrofurantoin）均表现出显著耐药性。9株普罗威登斯菌中有8株对壮观霉素（spectinomycin）耐药，6株对氯霉素（chloramphenicol）耐药，5株对新霉素（neomycin）耐药。所有12株分离菌株均对萘啶酸（nalidixic acid）、链霉素（streptomycin）、诺氟沙星（norfloxacin）、氨曲南（aztreonam）、西哌西林（cipericillin）、哌哌西林（pipericillin）以及头孢噻肟（cefotaxime）敏感；除OF008（摩根摩根菌（Morganella morganii））外，其余菌株均对氨苄西林（ampicillin）和头孢西丁（cefoxitin）敏感。油蝇肠道细菌的多重耐药性并非由突变率升高所介导。针对每株细菌，研究者分别测定了每10^8个细胞中利福平（rifampin）、萘啶酸（nalidixic acid）和壮观霉素（spectinomycin）平板上的耐药突变体数量。结果显示，各类耐药菌落的平均出现频率均比已知突变株ECOR 48低至少50倍。此外，油蝇肠道细菌未分泌抗菌物质。测试表明，所有油蝇肠道细菌均未在铜绿假单胞菌（Pseudomonas aeruginosa）、大肠埃希菌（Escherichia coli）、金黄色葡萄球菌（Staphylococcus aureus）以及白色假丝酵母（Candida albicans）的培养物上产生可检测到的抑菌圈。进一步研究发现，油蝇肠道细菌的耐药特性不仅针对抗生素，同样对有机溶剂具有耐受性。当预先用每毫升20微克的四环素处理后，9株油蝇肠道细菌中有7株可耐受100%环己烷（cyclohexane）覆盖；6株可耐受10%二甲苯（xylene）、苯或甲苯（toluene）（以90%环己烷稀释）；其中3株（OF007、OF010与OF011）可耐受50%二甲苯-50%环己烷混合液覆盖。观察到的抗生素耐药性与有机溶剂耐受性之间的相关性，可能由油蝇肠道细菌中存在的主动外排泵所介导，而该外排泵因兰乔拉布雷亚富含溶剂的环境所带来的持续选择压力得以保留。我们推测，油蝇肠道细菌及其携带的溶剂耐受相关基因，可能构成了抗生素耐药基因的微生物储库。