Antibiotic Exposure in a Low-Income Country: Screening Urine Samples for Presence of Antibiotics and Antibiotic Resistance in Coagulase Negative Staphylococcal Contaminants

Development of antimicrobial resistance has been assigned to excess and misuse of antimicrobial agents. Staphylococci are part of the normal flora but are also potential pathogens that have become essentially resistant to many known antibiotics. Resistances in coagulase negative staphylococci (CoNS) are suggested to evolve due to positive selective pressure following antibiotic treatment. This study investigated the presence of the nine most commonly used antimicrobial agents in human urine from outpatients in two hospitals in Ghana in relation to CoNS resistance. Urine and CoNS were sampled (n = 246 and n = 96 respectively) from patients in two hospitals in Ghana. CoNS were identified using Gram staining, coagulase test, and MALDI-TOF/MS, and the antimicrobial susceptibility to 12 commonly used antimicrobials was determined by disk diffusion. Moreover an analytical method was developed for the determination of the nine most commonly used antimicrobial agents in Ghana by using solid-phase extraction in combination with HPLC-MS/MS using electron spray ionization. The highest frequency of resistance to CoNS was observed for penicillin V (98%), trimethoprim (67%), and tetracycline (63%). S. haemolyticus was the most common isolate (75%), followed by S. epidermidis (13%) and S. hominis (6%). S. haemolyticus was also the species displaying the highest resistance prevalence (82%). 69% of the isolated CoNS were multiple drug resistant (≧4 antibiotics) and 45% of the CoNS were methicillin resistant. Antimicrobial agents were detected in 64% of the analysed urine samples (n = 121) where the most frequently detected antimicrobials were ciprofloxacin (30%), trimethoprim (27%), and metronidazole (17%). The major findings of this study was that the prevalence of detected antimicrobials in urine was more frequent than the use reported by the patients and the prevalence of resistant S. haemolyticus was more frequent than other resistant CoNS species when antimicrobial agents were detected in the urine.抗菌药物耐药性的产生被归因于抗菌药物的过量使用与滥用。葡萄球菌（Staphylococci）是人体正常菌群的组成部分，同时也是潜在致病菌，目前已对绝大多数已知抗生素产生耐药性。据推测，凝固酶阴性葡萄球菌（coagulase negative staphylococci, CoNS）的耐药性是抗生素治疗后产生正向选择压力而演化形成的。本研究针对加纳两家医院门诊患者的尿液样本，检测其中9种最常用抗菌药物的存在情况，并分析其与CoNS耐药性的关联。研究从加纳两家医院的患者中共采集尿液样本246份、CoNS菌株96株。本研究采用革兰氏染色（Gram staining）、凝固酶试验（coagulase test）以及基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）对CoNS进行鉴定，并通过纸片扩散法（disk diffusion）测定菌株对12种常用抗菌药物的药敏性。此外，本研究还建立了一种结合固相萃取（solid-phase extraction）与电喷雾电离（electron spray ionization）的高效液相色谱-串联质谱（HPLC-MS/MS）分析方法，用于检测加纳地区最常用的9种抗菌药物。结果显示，CoNS对青霉素V（penicillin V）、甲氧苄啶（trimethoprim）以及四环素（tetracycline）的耐药率最高，分别为98%、67%和63%。溶血葡萄球菌（Staphylococcus haemolyticus, S. haemolyticus）是最常见的分离菌株（占比75%），其次为表皮葡萄球菌（Staphylococcus epidermidis, S. epidermidis）（13%）和人葡萄球菌（Staphylococcus hominis, S. hominis）（6%）。溶血葡萄球菌同时也是耐药率最高的菌株种类，耐药率达82%。分离得到的CoNS中，69%为多重耐药菌株（耐≥4种抗菌药物），45%为耐甲氧西林菌株。经检测，64%的尿液样本（共121份）中可检出抗菌药物，其中检出率最高的为环丙沙星（ciprofloxacin，30%）、甲氧苄啶（trimethoprim，27%）以及甲硝唑（metronidazole，17%）。本研究的核心发现包括：尿液中抗菌药物的检出率高于患者自述的用药情况；且当尿液中检出抗菌药物时，耐药溶血葡萄球菌的检出率高于其他耐药CoNS菌株。