Emergence of linezolid resistance in a clinical Staphylococcus capitis isolate from Jiangsu Province of China in 2012
Linezolid (LZD) is an oxazolidinone antibacterial agent approved by FDA of America in the year of 2000 for the treatment of infection with gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (1), glycopeptide-intermediate S. aureus and vancomycin-resistant Enterococci. It exerts its antibacterial activity by acting on the early stage of the protein synthesis process; it binds to the ribosomal 23S portion of the 50S subunt of target bacteria and thereby inhibits the formation of the 70S initiation complex (2). LZD resistance occurs by mutations in the LZD 23S rRNA binding site, the ribosomal proteins L3 and/or L4 of the peptide translocation centre of the ribosome or by acquisition of a plasmid-borne ribosomal methyltransferase gene, cfr (3,4). In 2001, the first LZD-resistant S. aureus was reported in a US patient who had received a 1 month LZD treatment for dialysis-associated peritonitis (5). Since then, cases of LZD-resistant Staphylococcus have been reported worldwide in America (5-7), Europe (8-10) and Asia (11,12). In China, LZD was approved into clinical use in 2007 and several clinical cases of LZD-resistant Staphylococcus capitis have emerged in Zhejiang Province and the city of Beijing (13,14), but this is the first report, to our knowledge, of LZD-resistant Staphylococcus capitis in Jiangsu of China. The strain was isolated from blood sample of a patient with severe pulmonary infection. It showed an apparent resistance to LZD and was proved to have C2190T and C2561Y point mutations in the 23S rRNA and carry the cfr gene.
In August 2012, a 92-year old female who suffered recurrent pulmonary infections in recent two years was admitted to the geriatrics department because of the onset of cough and sputum for two days. The following CT scan showed that she got pneumonia (Figure 1). She was started with ceftazidime on an empirical treatment with moxifloxacin but showed no response to the treatment and developed a fever (39.0 °C) two days after admission. The subsequent bacterial cultures showed the coexistence of Pseudomonas aeruginosa, Acinetobacter baumannii and Aspergillus in her sputum sample and Enterococcus avium in the urine sample. Then we used diflucan to resist the fungal and ceftazidime, cefoperazone/sulbactam, imipenem, fosfomycin and tigecycline successively to combat the bacterial infection. To resist the gram-positive cocci, LZD was used on the 20th day after the patient’s admission with a dosage of 1,200 mg/d, and then the drug was discontinued 11 days later. Though the count of white blood cells (WBC) came to a transient decrease (drop from 19.5×109/L to 11.8×109/L) after the use of the antibiotics above, the patient eventually died of a sudden onset of ventricular tachycardia on the 35th day after admission. Patient in this case had long-term hospitalization history because of her recurrent pulmonary infection and had accepted lots antibiotics treatments. During her last hospitalization, the patient accepted airway intubation because of typeII respiratory failure and femoral vein catheterization to conduct hemodialysis for her renal failure. Because of her continued fever (varied between 37.0 and 39.0 °C), we conducted bilateral double bottles for blood cultures repeatedly to confirm the existence of bacteremia. We performed blood culture for three times, the first two (sampling on the 15th and 21th days after admission, respectively) both showed negative results whereas the last one (sampling on the 28th day after admission) showed a positive result. A strain of Staphylococcus capitis was isolated in last blood culture by using Viteck 2 compact (Figure 2A,B), and the strain showed resistance to LZD using the K-B method (Figure 2C) and also show high resistance with a MIC of 64 μg/mL using the broth dilution method. The Staphylococcus capitis also showed resistance to penicillin, pipercillin/tazobactam, cefepime, amikacin, levofloxacin, clindamycin while it was susceptible to cotrimoxazole, vancomycin and teicoplanin. Antimicrobial sensitivity test was conducted by the K-B disc diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (15). Domain V region of the 23S rRNA gene spanning 2001 to 2597bp (E. coli numbering) was amplified. Oligonucleotide premiers 5'-TGG GCA CTG TCT CAA CGA-3' and 5'-GGA TAG GGA CCG AAC TGT CTC-3' were used to amplify a 596bp fragment. Polymerase chain reaction (PCR) conditions were 30 cycles consisting of 94 °C for 1 min, for 30 s at 50 °C sec, and 72 °C for 1 min. The PCR fragments (596 bp) were purified and sequenced and the strain was found to have C2190T and C2561Y point mutations in the 23S rRNA. We also confirmed the existence of cfr gene in the Staphylococcus capitis using the method of PCR which was described in previous reports (16,17).
LZD is used for the treatment of infection with gram-positive bacteria, and had curative effect constantly since it was approved in 2000. Data from the USA and global surveillance studies report of LZD resistance were Staphylococcus aureus and 2% of coagulase-negative Staphylococcus (10,18-21).
A systematic review has shown that the mean time of LZD therapy reported prior to isolation of LZD-resistance coagulase-negative was 20 months, significant longer than case of LZD-resistance Staphylococcus (11 days) (22). In our case, the LZD resistance Staphylococcus capitis was isolated from the patient’s blood sample only eight days after the use of LZD, which suggested that short-term use of LZD may also induce the LZD-resistance coagulase-negative Staphylococcus. The cfr gene which is capable of transmitting horizontally between species is also an important factor leading to the LZD resistance. So we suggest that effective infection control measures should be enhanced to prevent the spread of multi-drug resistant strains.
The LZD resistance S. capitis in this case was isolated from a blood sample, which was documented in the study of Gu B et al. (22) that the most common samples cultured the LZD resistance coagulase-negative Staphylococcus. So when patients undergo conditions similar to this case: suffering a symptom of high fever, receiving invasive operations such as airway intubation, deep vein catheterization and so on, it is important for the clinicians to conduct blood culture to confirm whether there is a Staphylococcus induced blood stream infection. Timely detection of LZD resistance Staphylococcus is of great significance in the rational use of antibiotics and avoiding the emergence of multi-drug resistance bacteria.
Regarding to the mechanism of LZD resistance, the main explanation is mutations in the domain V region of the 23S rRNA gene. While the C2190T mutation was reported in several isolates of S. homin (23), this is the first report, to our knowledge, of C2190T and C2561Y mutations in Staphylococcus capitis. The isolate was also PCR-positive for the cfr, a gene located on a transferable element, which indicated a potential to disseminate horizontally among Gram-positive pathogenic strains.
This article systematically reviews the published literature for case reports of LZD-resistant coagulase-negative Staphylococcus (LRCoNS) (Table 1). In all reported cases, strains including S. cohnii, S. epidermidis, S. lugdunensis, S. hominis and S. kloosii were isolated from aseptic sample, which included blood, pus, bronchoalveolar lavage fluid. Nevertheless blood sample was the vast majority. LRCoNS were reported worldwide, including North America, South America, European and Asia. All these strains reported were conducted the susceptibility test by broth microdilution. The mechanisms for LZD resistance were L3/L4 mutation, G2603T, T2504A, A2503 mutations in the 23S rRNA and the presence of a transmissible cfr ribosomal methyltransferase. The outcome of the LRCoNS infected patients is obscure, however, two cases mentioned the patients were died in the end, which had the same outcome in our report.
In conclusion, though the LZD-resistant Staphylococcus is still sporadic now, the prolonged hospital stays, frequent interventions and abuse of antibiotics may accelerate the dissemination of LZD resistance Staphylococcus. Judicious use of LZD and surveillance of resistance in staphylococci are necessary to preserve the therapeutic efficacy of this important antimicrobial.
Disclosure: The authors declare no conflict of interest.
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