In vitro susceptibility to methicillin, vancomycin and linezolid of staphylococci isolated from bloodstream infections in eastern Turkey.

Staphylococcus species are one of the major causes of bacterial bloodstream infections. Multi-resistant staphylococci infections are major therapeutic problems. This study was aimed to detect methicillin, linezolid and vancomycin susceptibilities of Staphylococcus isolates. A total of 870 Staphylococcus strains isolated from blood cultures of hospitalized patients with BSI. Antimicrobial susceptibilities of methicillin, linezolid and vancomycin were detected according to the Clinical and Laboratory Standards Institute (CLSI). A total of 771 (88.6%) isolates were coagulase-negative staphylococci (CoNS). 700 (80.5%) isolates were methicillin-resistant (MR) and 170 (19.5%) were methicillin-susceptible (MS). All the MS isolates were also susceptible to linezolid. However 15 (1.7%) of MR strains were resistant to linezolid. The minimum inhibitory concentration range for the linezolid-resistant isolates by Etest was 6-32 μg/mL. The difference between linezolid susceptibilities for MS and MR staphylococci was not quite statistically significant (p = 0.052). There was no statistically significant difference between S. aureus and CoNS isolates for linezolid susceptibility. All of the isolates were susceptible to vancomycin. In conclusion, linezolid is currently an efficient option for the treatment of methicillin-resistant staphylococci infections.

Bacterial bloodstream infections (BSI) are important problems worldwide. Staphylococcus species are one of the major causes of BSI, in both community- and hospital-acquired diseases. Staphylococci are widespread in nature, also they are commensal microorganisms located on the surface of the human skin and mucous membranes (Bannerman, 2003; Winn ; Yok-Ai Que and Moreillon, 2010). Staphylococcus aureus and coagulase-negative staphylococci (CoNS), mainly Staphylococcus epidermidis, are the most common bacteria isolated from blood (Bannerman, 2003; Natoli ; Winn ). In addition, Yok-Ai Que and Moreillon (2010) stated that CoNS may adhere to medical devices and hospital environmental surfaces by forming bio-films. These bacteria can easily spread from person to person through patient fluids, contaminated medical devices and on the hands of health-care workers (Bannerman, 2003; Yok-Ai Que and Moreillon, 2010). The isolation rate of S. aureus from BSIs has universally increased. Wisplinghoff reported that CoNS and S. aureus were responsible for approximately 30% and 20% of the nosocomial BSIs, respectively.

In recent years, infections caused by multi-resistant staphylococci have become one of the major therapeutic problems (Deveci ). The emergence of these multidrug resistant isolates is often attributed to long term usage of indwelling central venous catheters, administration of parenteral nutrition, long term usage of broad-spectrum antibiotics and increasing number of immunocompromised patients (Winn ; Yok-Ai Que and Moreillon, 2010). These problematic pathogens have acquired resistance to methicillin and some of which to many non-beta-lactam antibiotics (Boyle-Vavra and Daum, 2007; Niemeyer ; Winn ). Methicillin resistance in staphylococci is caused by expression of the penicillin-binding protein 2a (PBP2a) encoded by the mecA gene which is located in the staphylococcal cassette chromosome mec (SCCmec) and can be transmitted via horizontal transfer between Staphylococcus species (Boyle-Vavra and Daum, 2007; Niemeyer ; Yok-Ai Que and Moreillon, 2010). Currently, over 80% of clinical Staphylococcus isolates throughout the world are resistant to penicillin due to β-lactamase production (Winn ). Until recently, glycopeptides such as vancomycin were the first choice drug to treat infections caused by methicillin-resistant staphylococci (Yok-Ai Que and Moreillon, 2010). However, vancomycin-resistant Staphylococcus isolates have been detected in various countries (Centers for Disease Control and Prevention (CDC), 1997, 1999, 2002, 2004; Gemmell ; Hiramatsu ; Palazzo ).

Linezolid was introduced by Ford as a member of the oxazolidinone class of antibiotics. This antibiotic demonstrates potent antimicrobial activity against most multi-resistant Gram-positive microorganisms, including methicillin-resistant coagulase-negative staphylococci (MRCoNS), methicillin-resistant S. aureus (MRSA), multidrug-resistant (MDR) Streptococcus pneumoniae and vancomycin-resistant enterococci (VRE) (Ford ). Linezolid inhibits bacterial protein syntheses by binding to the domain V region of 23S rRNA at an early step, and prevents the formation of the N-formylmethionyl-tRNA-mRNA-70S ribosomal tertiary complex. Mutations in the central loop of the domain V region are the most frequent causes of linezolid resistance (Khan ; Kloss ).

This study presented here aimed to detect methicillin, linezolid and vancomycin susceptibilities for CoNS and S. aureus isolates collected from blood cultures in Dicle University Hospital, Diyarbakir-Turkey.

A total of 870 staphylococcal isolates collected from blood cultures of hospitalized patients in Dicle University Hospital between January 2007 and August 2011 were included retrospectively in this study. Dicle University Hospital is a tertiary care center with capacity of 1400 beds. Among 870 staphylococcal isolates included in this study, 442 were collected from adult patients (ranged 17–65 years), 237 from pediatric patients (≤ 16 years) and 191 from newborn babies admitted into the neonatal intensive care unit (NICU). Only isolates from clinically significant BSI (one isolate per patient) were included in this study. Clinically significant BSI can be diagnosed when two or more of the following criteria are present with positive blood cultures taken from both arms: -body temperature ≤ 36 °C or ≥ 38 °C;-heart rate > 90 beats/min; -respiratory rate > 20 breaths/min or an arterial partial pressure of carbon dioxide (PaCO2) < 4.3 kPa (32 mmHg); -leukocytes < 4,000 cells/mm3 (4×109 cells/L) or > 12,000 cells/mm3 (12×109 cells/L) or > 10% immature neutrophils (band forms) (American college of chest physicians / society of critical care medicine consensus conference, 1992). Blood culture bottles were incubated in Bactec™ BD 9120 and 9240 (Becton Dickinson, MD, USA) automated blood culture systems at 37°C for 7–10 days. After growth, the culture was inoculated onto 5% sheep blood agar (Oxoid Ltd., Basingstoke, UK) and the plate incubated at 35 ± 2 °C for 18–24 h. Isolate identification was performed by routine methods of Gram staining, catalase activity, slide and tube coagulase tests, DNAse test, and also BD Phoenix™ 100 (Becton Dickinson, MD, USA) the fully automated microbiology system by using the manufacturers’ protocol.

Antimicrobial susceptibility testing for methicillin and linezolid were performed by Kirby-Bauer’s disk diffusion method and BD Phoenix™ 100 the fully automated microbiology system by using the manufacturers’ protocol according to the recommendations of Clinical and Laboratory Standards Institute (CLSI) (2009). Methicillin susceptibility was investigated with 1 μg oxacillin and 30 μg cefoxitin disk (Oxoid Ltd., Basingstoke, UK). Linezolid susceptibility was tested using 30 μg linezolid disk (Oxoid Ltd., Basingstoke, UK) and linezolid resistance was confirmed by Etest strips (bioMerieux SA, Marcy l’Etoile, France) (Abb, 2002; CLSI, 2009). In addition, vancomycin susceptibility was also investigated using Etest strips and the fully automated microbiology system according to the CLSI breakpoints (2009) For disk diffusion method and Etest strips, a 0.5 McFarland standard suspension was inoculated onto Mueller-Hinton agar (Merck KGaA, Darmstadt, Germany) plates as described by CLSI (2009). MHA plates were incubated at 35 ± 2 °C for 24 hours. Inhibition zone diameter was measured at 24 hours in transmitted light for linezolid.

For methicillin susceptibility, in accordance with CLSI guidelines, inhibition zone diameter for oxacillin ≥ 13 mm was considered as susceptible, 11–12 mm as intermediate, ≤ 10 mm as resistant; for cefoxitin ≥ 22 mm was considered as susceptible, ≤ 21 mm as resistant for S. aureus and S. lugdunensis, ≥ 25 mm was considered as susceptible, ≤ 24 mm as resistant for the other CoNS except S. lugdunensis. Inhibition zone diameter for linezolid ≥ 21 mm was considered as susceptible, < 21 mm as resistant. MIC value for linezolid ≤ 4 μg/mL was considered as susceptible, > 4 μg/mL as resistant. According to the MIC interpretive standard of CLSI, MIC value for vancomycin ≥ 2 μg/mL was considered as susceptible, 4–8 μg/mL as intermediate, ≥ 16 μg/mL as resistant.

S. aureus ATCC 25923 was used for quality control in the fully automated microbiology system, Etest strips and disk diffusion method. Data were analyzed by Epi Info™ 7-Community Edition (Centers for Diseases Control and Prevention, Atlanta, GA, USA) statistical package program. Statistical evaluation between the groups of methicillin-susceptible and -resistant isolates for linezolid susceptibility was performed with the Fisher’s exact test. Also, statistical evaluation between S. aureus and CoNS isolates for linezolid susceptibility was analyzed with the chi-square test. The p-value of < 0.05 was selected for statistical significance.

In the present study, 771 (88.6%) of 870 isolates were coagulase-negative staphylococci (CoNS), and 99 (11.4%) S. aureus. According to the data from the antimicrobial susceptibility testing, 700 (80.5%) isolates were methicillin-resistant staphylococci (MRS), and 170 (19.5%) methicillin-susceptible staphylococci (MSS). All the MSS isolates were also susceptible to linezolid. However 15 (1.7%) of MRS strains were resistant to linezolid. The MIC range for the linezolid-resistant isolates using Etest was 6–32 μg/mL. In addition, the MIC value for all the linezolid-resistant isolates using the fully automated microbiology system was recorded as > 4 μg/mL uniformly. The difference between linezolid susceptibilities for MSS and MRS was not quite statistically significant (p = 0.052). Among 15 methicillin- and linezolid-resistant staphylococcal isolates, only one was S. aureus and the others were CoNS (≤). There was no statistically significant difference between S. aureus and CoNS isolates for linezolid susceptibility. All staphylococci tested were susceptible to vancomycin according to the both Etest and the fully automated microbiology system. The MIC value for all the vancomycin susceptible isolates using the both Etest and the fully automated microbiology system was recorded as ≤ 2 μg/mL uniformly.

CoNS and S. aureus are frequent causes of bacteremia and sepsis, in both community and hospitalized patients (Natoli ; Winn ). In recent years, due to the increasing antimicrobial resistance, treatment of staphylococcal infections has become more challenging. Currently, over 80% of clinical staphylococcal isolates are resistant to penicillin worldwide. In addition over 60% of nosocomial S. aureus and CoNS isolates are resistant to methicillin in some Asian countries including China and Turkey. Lin stated methicillin-resistant staphylococci as also cross-resistant to other β-lactams and to a wide range of other antibiotics (22). In our study 80.5% of total 870 isolates were MRS. Therefore, beta-lactams are inefficacious agents to treat staphylococcal infections in our hospital.

Until recently, most Staphylococcus isolates were susceptible to glycopeptides such as vancomycin. The first cases of infections with glycopeptides-resistant S. haemolyticus were reported in 1986 and the first VISA isolate was described in Japan (Hiramatsu ). Shortly after, Staphylococcus isolates with reduced susceptibility to vancomycin, and with intermediate and full resistance to this drug were also reported in many countries (Bataineh, 2006; Howe, 1998; Palazzo, 2005). At the beginning of the second millennium, eight cases of vancomycin-intermediate S. aureus (VISA) and one of vancomycin-resistant S. aureus (VRSA) (MIC value for vancomycin ≥ 32 μg/mL) were documented in the United States (CDC, 2002). Palazzo reported vancomycin-resistant staphylococcal isolates in healthy carriers that have not received antibiotic treatment.

In Turkey, some isolates showing reduced susceptibility or intermediate resistance for vancomycin had also been reported. Torun reported two heterogeneously vancomycin-intermediate clinical isolates of methicillin-susceptible and methicillin-resistant S. aureus. Sancak have also documented a MRSA isolate displaying heterogeneous resistance to vancomycin in a Turkish university hospital. Despite that, there was no report of vancomycin full resistance among staphylococcal isolates in Turkey. In the present study, all staphylococci analyzed were susceptible to vancomycin. Indeed, VISA isolates could not be detected by the susceptibility testing methods used. The fact that we have used the fully automated microbiology system could have influenced the VISA negative detection. However, we have also included the Etest for determining the MIC value for vancomycin. MIC determinations by agar or broth dilution, in addition to Etest using a 0.5 McFarland turbidity standard, have been recommended for detecting VISA isolates (Sader ). The only observation is that in this case susceptibility breakpoint should be adjusted to ≤ 1 μg/mL. Sader have suggested that the susceptibility breakpoint be modified to ≤ 1 μg/mL.

In the present study we found that only 1.7% of total 870 staphylococcal isolates was resistant to linezolid according to the both disk diffusion method and the fully automated microbiology system. Linezolid resistance was confirmed by using Etest strips in this study. Many researchers have accepted that the Etest method is credible method to detect the MIC value (Bell ; CLSI, 2009; Kaya et al., 2009; Thool ). But CLSI (2009) recommended the confirmation of resistance to linezolid by using agar or broth dilutions. In addition, Gemmell said that the mean Etest MIC was approximately one two-fold dilution lower than the mean microdilution MIC. Also Tenover reported that further studies of the agar-based methods (disk diffusion method and Etest) are needed to better define the optimal endpoints for interpreting results of testing for linezolid against staphylococci and enterococci. Arias stated that disk diffusion susceptibility tests or Etest might not detect cfr-mediated linezolid resistance when standard procedures are used and that a longer time of incubation may be needed.

Thus, linezolid is still an effective agent for staphylococcal isolates obtained from blood culture in our hospital. Data from LEADER surveillance program in USA, from linezolid program (ZyvoxR Annual Appraisal of Potency and Spectrum; ZAAPS) in European countries and from many studies in Spain and in our country found that linezolid sensitivity rates in staphylococci were approximately 99% (Jones ; Ross ). It is important to remark that five linezolid-resistant S. kloosii isolates were obtained from hospitalized patients in the plastic surgery (one isolate), pediatrics clinic (one isolate) and neonatal intensive care unit (NICU; three isolates). This result seems to indicate the occurrence of S. kloosii small outbreak in the NICU of the studied hospital. Recently, Peer reported a case of sepsis with an intracranial bleed in a 60-year-old male from whom linezolid-resistant S. kloosii was repeatedly isolated from blood cultures, demonstrating the potential of this staphylococcal species to cause BSI in immunocompromised host. S. epidermidis was not detected among the linezolid-resistant isolates in the present study. However, it is possible that the fully automated identification system used may have failed to detect some of the resistant isolates.

Due to the emergence of vancomycin-intermediate and -resistant isolates, poor tissue penetration, and the fact that vancomycin must be given intravenously, new therapeutic options are necessary for the treatment of staphylococcal infections. Our data showed that, in our hospital, linezolid can be a therapeutic option for treatment of infections cause by both MRCoNS and MRSA. Linezolid is a well-tolerated agent currently available in oral and intravenous forms. The oral form is 100% bio-available and the earlier switching from intravenous to oral therapy is possible (Weigelt ). In a cohort study of Lodise , the incidence of nephrotoxicity with higher daily (≥ 4 g/day) and lower daily doses of vancomycin < 4 g/day) were compared with nephrotoxicity of linezolid. According to this study, nephrotoxicity was significantly higher in the high-dose vancomycin group (34.6%) when compared with the low-dose vancomycin group (10.9%) or with the linezolid group (6.7%).

In conclusion, the treatment of methicillin-resistant staphylococcal infections has been a growing problem in both hospital- and community-acquired diseases. For this reason, alternative antimicrobial agents with anti-staphylococcal activity are needed. The results presented here are in line with data of previous national and international investigations suggesting that linezolid is an efficient option for the treatment of a number of infections caused by methicillin-resistant staphylococci (Lin ; Weigelt ; Zorgani ). Finally, due to the increasing antimicrobial resistance among staphylococcal isolates, resistance rates should be reported periodically.

Table 1

Bacterial species and hospital setting for methicillin-linezolid resistant staphylococcal isolates.

Isolate no	Species	Inpatient Clinics
DUH1	S. kloosii	Plastic Surgery
DUH2	S. aureus	Nephrology
DUH3	S. hyicus	Pediatric Infectious Diseases
DUH4	S. cohnii	Oncology
DUH5	S. cohnii	Chest Diseases
DUH6	S. kloosii	Neonatal Intensive Care Unit
DUH7	S. cohnii	Cardiology
DUH8	S. kloosii	Neonatal Intensive Care Unit
DUH9	S. schleiferi	Neonatal Intensive Care Unit
DUH10	S. kloosii	Pediatric Infectious Diseases
DUH11	S. capitis	Neonatal Intensive Care Unit
DUH12	S. kloosii	Neonatal Intensive Care Unit
DUH13	S. capitis	Endocrinology
DUH14	S. cohnii	Adult Infectious Disease
DUH15	S. hominis	Hematology