Abstract
Background
Staphylococcus aureus and other Staphylococcus species are important pathogenic organisms and are responsible for various hospital infections. These are the predominant organisms found in pus and blood culture isolates. Infections arising due to these bacterial isolates are difficult to treat because of developing multidrug resistance.
Methods
Over a 1-year period at a tertiary care hospital laboratory, 524 Staphylococci species were isolated from pus, blood and urine samples and species-level identification was done.
Results
S. aureus formed the predominant species (70.8%) followed by coagulase-negative Staphylococcus (CoNS) (29.20%). S. aureus (91%) was the main isolate from pus samples; however, CoNS was isolated in equally higher proportion in blood culture (63.58%). Among the CoNS, Staphylococcus hemolyticus was the main isolate (9.3%). β-Lactamase production, alteration of PBP and MLSB resistance were seen in variable degrees in different species.
Conclusion
CoNS group of Staphylococci is becoming an important cause of infection at tertiary care centres. The increased multidrug resistance among various Staphylococcus species is a cause of great concern and requires adequate measures to prevent the spread of these microorganisms in the hospital and the community.
Keywords: Staphylococcus aureus, Coagulase-negative Staphylococcus, Antibiotic resistance, Macrolide–lincosamide–streptogramin-B antibiotic resistance
Introduction
Staphylococci are among the most important bacteria that cause diseases in humans. The coagulase-positive Staphylococcus aureus is the most important human pathogen in this genus. Coagulase-negative Staphylococcus (CoNS) have increasingly been associated as opportunistic pathogens with serious nosocomial infections.1 Isolates of S. aureus that have become resistant to methicillin are known as methicillin-resistant S. aureus (MRSA). The infection acquired by persons who have neither been hospitalized nor undergone any medical procedure is referred to as ‘Community-acquired’ (CA-MRSA), and when acquired in the hospitalized patient, it is referred as hospital-acquired (HA-MRSA).2
Antimicrobial therapy is vital to the management of patients having staphylococcal infections. Antibiotic sensitivity pattern of such clinical isolates is becoming unpredictable and requires testing as a guide to therapy.3 Since the 1990s, CA-MRSA infections have been reported from various countries. CoNS are part of normal skin flora and have emerged as important pathogens in hospital-acquired infections. However, these have been usually sensitive to commonly used antibiotics till a decade back.
Due to increasing resistance to antimicrobial agents among Staphylococci, renewed interest has emerged to the use of macrolide (erythromycin, clarithromycin, roxithromycin and azithromycin), lincosamide (clindamycin and lincomycin) and streptogramins (streptogramin A – pristinamycin and dalfoprisin; and streptogramin B – quinupristin). However, development of macrolide resistance worldwide has limited the use of these antibiotics. Macrolide resistance occurs either through target site modification (MLSB phenotype, encoded by erm genes), efflux pump mechanism (MS phenotype, i.e. resistant to macrolide and streptogramins but sensitive to lincosamide, encoded by msrA/B genes) or decreased cell wall permeability. By the use of ‘D test’, inducible (iMLSB) and constitutive (cMLSB) can be differentiated. When the ‘D test’ is positive, it is iMLSB, and when the resistance is both towards clindamycin and to erythromycin, it is cMLSB. In vitro antibiotic sensitivity tests normally done in the laboratory cannot detect inducible resistance unless the ‘D test’ is done. Constitutive resistance to MLS antibiotics is confirmed by using molecular methods for the concerned erm genes. The msrA/B gene, first identified in Staphylococcus epidermidis, confers the so-called MS phenotype as ascribed to earlier. The msrA/B genes may be found in Staphylococcus aureus but are more common in CoNS.
On admission to hospital and treatment with antibiotics, patients often become colonised with more drug-resistant S. aureus and CoNS. In the recent past, CoNS has gained more clinical significance as they have been isolated in more numbers from patients having various risk factors, e.g. use of various intravascular catheters, prosthetic devices, foreign body implants, use of immunosuppressive drugs for renal transplant recipients and immunocompromised patients on chemotherapeutic agents. These infections are difficult to treat because of the underlying risk factors and increased drug resistance among CoNS species. The present study was carried out to identify the frequency of various Staphylococcus species and their current antibiogram pattern.
Materials and methods
A total of 524 isolates of Staphylococci were collected from pus, blood, urine and other miscellaneous samples including body fluids and sputum over a 1-year period from November 2012 to October 2013 at the laboratory of a tertiary care hospital and studied for antibiotic sensitivity patterns. The isolates were considered relevant when isolated in pure culture from infected sites. These isolates were initially identified by colony morphology, Gram staining, catalase, slide and tube coagulase tests and anaerobic acid formation from mannitol.3 Further identification of different species of CoNS and antibiotic susceptibility was done by VITEK2 system (Biomerieux). The system identified beta-lactamases and PBP2 resistance. Kirby–Bauer disc diffusion method uses panel of required antibiotics as per CLSI (2013) guidelines that were placed in parallel.4 Discs contained the following antibiotics at specific absolute concentrations – penicillin (10 μg), oxacillin (20 μg), linezolid (30 μg), cefoxitin (30 μg), augmentin (20/10 μg), clindamycin (2 μg), erythromycin (15 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), tetracycline (30 μg) and gentamicin (10 μg).
Data were generated for β-lactamase production, modification of PBP2 and MLSB resistance (both inducible and constitutive). The phenotypic method was used for our study by using erythromycin (15 μg) and clindamycin (2 μg) discs, kept 15 mm apart, for inducible type of MLSB resistance. Isolates were considered having inducible resistance to clindamycin when showing flattening of clindamycin sensitivity zone adjacent to erythromycin sensitivity zone. Novobiocin resistance tests were also placed for quality control and confirmation. Controls ATCC 25923 for MRSA and 29213 for MSSA were used. Only phenotypic methods have been used in our study to distinguish the different isolates of Staphylococcus aureus (MRSA and MSSA) and coagulase-negative Staphylococci (CoNS).
Results
S. aureus was the single most common isolate obtained from all the clinical samples (70.8%) and was the predominant isolate from pus (91.8%). CoNS were the predominant microorganisms from blood samples (63.58%). Among the 10 identified CoNS species, S. hemolyticus was the most frequently isolated (9.3%), followed by S. epidermidis (8%) and S. saprophyticus (3.6%). From blood samples, S. hemolyticus (19.6%) and S. epidermidis (17.8%) were the main isolates, followed by S. hominis (6.08%) and S. saprophyticus (5.4%).
From urine, the main CoNS isolates were S. saprophyticus (22.8%) and S. epidermidis (20%). Detailed breakdown of isolates is given in Table 1. Antibiotic susceptibility testing showed varying degree of resistance by different staphylococcal species (Table 2). Maximum resistance was observed to penicillin, oxacillin and ciprofloxacin. S. aureus showed higher resistance to penicillin (80%), oxacillin (53%), augmentin (54%) and ciprofloxacin (85%). Thus, there were 153 CoNS isolates, and out of 371 isolates of S. aureus, 196 were MRSA (53%) and 175 were MSSA (47%). S. hemolyticus showed maximum resistance to penicillin (100%), oxacillin (100%) and augmentin (90%). All the strains of S. hemolyticus (100%) were β-lactamase producers, followed by S. epidermidis (89%) and S. aureus (80%). Resistance to MLSB (inducible) resistance was observed in 17.74% of the staphylococcal isolates and 10.30% had the MLSB (constitutive) type. S. aureus had maximum resistance at 19% (Table 3).
Table 1.
Species-wise distribution of different Staphylococcus isolates.
| Species | Pus | Blood | Urine | Misc | Total |
|---|---|---|---|---|---|
| S. aureus | 280 | 54 | 21 | 16 | 371 |
| S. hemolyticus | 11 | 29 | 01 | 08 | 49 |
| S. epidermidis | 06 | 26 | 08 | 02 | 42 |
| S. saprophyticus | 01 | 08 | 09 | 01 | 19 |
| S. hominis | 02 | 09 | 0 | 02 | 13 |
| S. xylosus | 03 | 05 | 0 | 0 | 08 |
| S. warneri | 0 | 04 | 01 | 02 | 07 |
| S. scuiri | 01 | 03 | 0 | 0 | 04 |
| S. capitis | 0 | 04 | 0 | 0 | 04 |
| S. lentis | 0 | 04 | 0 | 0 | 04 |
| S. lugdunensis | 01 | 02 | 0 | 0 | 03 |
| Total | 305 | 148 | 40 | 31 | 524 |
Table 2.
Antibiotic resistance pattern (%) of main Staphylococcus species.
| Species | P | Ox | Aug | Clind | E | Cip | Levo | Lz | Tet | Tg | Genta | Cx |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| S. aureus | 296 | 196 | 201 | 92 | 92 | 316 | 86 | 0 | 15 | 0 | 119 | 196 |
| (371) | (80%) | (53%) | (54%) | (25%) | (25%) | (85%) | (23%) | (0%) | (4%) | (0%) | (32%) | (53%) |
| S. hemolyticus | 49 | 49 | 44 | 8 | 8 | 41 | 41 | 0 | 2 | 0 | 41 | 49 |
| (49) | (100%) | (100%) | (90%) | (17%) | (17%) | (84%) | (84%) | (0%) | (4%) | (0%) | (84%) | (100%) |
| S. epidermidis | 37 | 34 | 33 | 32 | 32 | 19 | 8 | 0 | 0 | 0 | 12 | 34 |
| (42) | (88%) | (81%) | (79%) | (77%) | (77%) | (45%) | (19%) | (0%) | (0%) | (0%) | (27%) | (81%) |
| S. saprophyticus | 14 | 7 | 14 | 8 | 7 | 9 | 3 | 0 | 4 | 3 | 8 | 7 |
| (19) | (74%) | (37%) | (74%) | (42%) | (37%) | (48%) | (16%) | (0%) | (22%) | (16%) | (42%) | (37%) |
| S. hominis | 9 | 9 | 10 | 10 | 11 | 5 | 5 | 9 | 3 | 1 | 0 | 9 |
| (13) | (69%) | (69%) | (77%) | (77%) | (85%) | (38%) | (38%) | (69%) | (23%) | (8%) | (0%) | (69%) |
| S. xylosus 2 | 2 | 2 | 2 | 2 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 2 |
| (8) | (25%) | (25%) | (25%) | (25%) | (0%) | (0%) | (0%) | (0%) | (13%) | (0%) | (0%) | (25%) |
| S. warneri | 5 | 4 | 4 | 2 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 4 |
| (7) | (72%) | (57%) | (57%) | (29%) | (14%) | (14%) | (14%) | (0%) | (0%) | (0%) | (0%) | (57%) |
| S. lentis | 2 | 1 | 2 | 3 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 3 |
| (4) | (50%) | (25%) | (50%) | (75%) | (0%) | (25%) | (25%) | (25%) | (25%) | (25%) | (25%) | (75%) |
| S. capitis | 1 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| (4) | (25%) | (25%) | (25%) | (25%) | (50%) | (0%) | (0%) | (0%) | (0%) | (0%) | (25%) | (25%) |
| S. sciuri | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 0 | 0 | 0 | 2 |
| (4) | (50%) | (50%) | (50%) | (50%) | (50%) | (50%) | (50%) | (25%) | (0%) | (0%) | (0%) | (50%) |
| S. lugdunensis | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| (3) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) | (0%) |
P, penicillin; Ox, oxacillin; Aug, augmentin; Clind, clindamicin; E, erythromycin; Cip, ciprofloxacin; Levo, levofloxacin; Lz, linezolid; Tet, tetracycline; Tg, tigecycline; Genta, gentamicin; Cx, cefoxitin.
Note: All percentages refer to % resistance. The total number of isolates is given below the name of the organism.
Table 3.
Species-wise drug resistance pattern in different Staphylococcus isolates.
| Species | β-Lactamase | PBP (mecA)/modification of PBP | iMLSB | cMLSB |
|---|---|---|---|---|
| S. aureus | 296 | 196 | 70 | 22 |
| (371) | (80%) | (53%) | (19%) | (6%) |
| S. hemolyticus | 49 | 49 | 02 | 06 |
| (49) | (100%) | (100%) | (4.0%) | (12%) |
| S. epidermidis | 37 | 34 | 16 | 16 |
| (42) | (89%) | (82%) | (16%) | (16%) |
| S. saprophyticus | 14 | 07 | 02 | 06 |
| (19) | (75%) | (36%) | (10%) | (31%) |
| S. hominis | 09 | 09 | 02 | 01 |
| (13) | (69%) | (69%) | (15%) | (8%) |
| S. xylosus | 02 | 02 | 0 | 02 |
| (8) | (25%) | (25%) | (25%) | |
| S. warneri | 05 | 04 | 0 | 01 |
| (7) | (71%) | (57%) | (14%) | |
| S. lentis | 02 | 01 | 0 | 0 |
| (4) | (50%) | (25%) | ||
| S. capitis | 01 | 01 | 01 | 0 |
| (4) | (25%) | (25%) | (25%) | |
| S. scuiri | 02 | 01 | 0 | 0 |
| (4) | (50%) | (25%) | ||
| S. lugdunensis | 0 | 0 | 0 | 0 |
| (3) |
PBP, penicillin-binding protein; MLSB, macrolide–lincosamide–streptogramin B; iMLSB, Inducible MLSB; cMLSB, constitutive MLSB.
The total number of isolates is given below the name of the organism.
Discussion
S. aureus is the most commonly isolated bacterial pathogen and may be considered to be an emerging epidemic.5 In our study, out of 524 isolates, S. aureus was the commonest organism, while CoNS constituted nearly one-third of the total isolates. Among the CoNS, S. hemolyticus was the commonest isolate followed by other species as given in Table 1. The most frequently isolated species of CoNS in past studies were S. epidermidis (up to 80%) and S. saprophyticus (15.60%).6, 7, 8 15% S. hemolyticus was isolated in another study.9 Frequency of different species varies considerably in different clinical samples, which is coherent with other studies.10, 11
In our study, maximum resistance to penicillin (79.58%) and oxacillin (58%) was observed. S. aureus showed 80% resistance against penicillin and 53% resistance against oxacillin. CoNS had similar resistance against penicillin (79%), but much higher resistance against oxacillin (71%). Older studies have shown more than 80% of CoNS isolates being resistant to methicillin and semisynthetic penicillins, and among them, S. hemolyticus showed maximum (100%) resistance to penicillin and oxacillin.10, 11, 12 In our study, S. hominis and S. epidermidis showed higher resistance to erythromycin (85% and 77%). Both these isolates also showed higher resistance to clindamycin (77%) similar to another study done elsewhere.9 S. hominis, S. lentis and S. sciuri showed resistance to linezolid (69%, 25% and 25%). In our study, S. aureus, S. hemolyticus and S. epidermidis showed higher resistance to ciprofloxacin (85%, 84% and 45%) as compared to levofloxacin (23%, 84% and 19%), while lower resistance was observed elsewhere.7 S. lugdunensis did not show any drug resistance. Higher sensitivity was seen to vancomycin (100%) followed by linezolid, which again is coherent with other studies.10, 11 S. hemolyticus showed maximum multidrug resistance to all the commonly used antibiotics.
Production of β-lactamase remains the single most common mechanism of drug resistance followed by modification of penicillin-binding proteins. Both these mechanisms are seen in all species in variable frequency except S. lugdunensis (Table 3). iMLSB type resistance is seen more frequently (17.74%) than cMLSB type (10.30%), but less frequently than the previous two mechanisms. S. xylosus and S. warneri showed resistance only of cMLSB type.
Most frequently isolated species of CoNS that were isolated from clinical samples in past studies are S. epidermidis (up to 80%) and S. saprophyticus (15.60%).6, 7, 8 Among CoNS, S. hemolyticus remained the commonest isolate (32%) followed by S. epidermidis (27.45%) in our study; 15% S. hemolyticus was reported in another study.9 S. hominis (8.4%), S. xylosus (5.2%) and S. warneri (4.5%) were considered as important isolates in our study, with S. lugdunensis having the least frequency (1.9%). This is much lower as compared with another study.9 It appears that frequency of different species varies on different locations and hospitals. S. saprophyticus was predominantly isolated from urine samples and this frequency is coherent with other studies.10, 11
We observed maximum resistance to penicillin and oxacillin. Older studies have shown more than 80% of CoNS isolates being resistant to methicillin and semisynthetic penicillins, and among them, S. hemolyticus showed maximum resistance to penicillin and oxacillin (up to 100%).10, 11, 12 S. hominis and S. epidermidis showed higher resistance to erythromycin (85% and 77%). Both these isolates also showed higher resistance to clindamycin (77%) similar to another study done elsewhere.9 S. hominis, S. lentis and S. sciuri showed resistance to linezolid (69%, 25% and 25%). S. aureus, S. hemolyticus and S. epidermidis showed higher resistance to ciprofloxacin (85%, 84% and 45%) as compared to levofloxacin (23%, 84% and19%) in our study, while lower resistance was observed elsewhere.7 S. lugdunensis did not show any drug resistance. Higher sensitivity was seen to vancomycin followed by linezolid, which again is coherent with other studies.10, 11
Production of β-lactamase remains the single most common mechanism of drug resistance followed by modification of penicillin-binding proteins. Both these mechanisms are seen in all species in variable frequency except S. lugdunensis (Table 3). iMLSB (17.74%) type resistance is seen more frequently than cMLSB type (10.30%), but less frequently than the previous two mechanisms. S. xylosus and S. warneri showed resistance only of cMLSB type. Our study further re-emphasises that the results of the double disc diffusion tests do correlate well with fully automated systems. Fiebelkorn et al. described this reliable method (double disc diffusion test) for detecting inducible resistance to clindamycin in erythromycin-resistant isolates of S. aureus and CoNS.12
We suggest that a simple test for iMLSB can be done by any laboratory to rule out inducible lincosamide resistance by carrying out the ‘D’ test. CoNS must be looked into carefully so that there are no treatment failures, and thus unnecessary clindamycin usage can be avoided as recommended elsewhere as well.4, 13 Where molecular tests can be done, the corresponding genes for constitutive resistance can also be mapped in selected cases. By adopting such an approach, clinicians with the support of microbiologists, through correct identification and an early alert for resistance to MLSB antibiotics, can help in reducing morbidity and perhaps, saving lives.
Conclusion
We conclude that S. aureus remains the most common pathogenic organism among all Staphylococcus species and CoNS are replacing S. aureus in blood and urine specimens. The frequency of CoNS species varies in different locations, regions and hospitals. In the present study, majority of CoNS belonged to S. hemolyticus and S. epidermidis, and this increase in frequency of S. hemolyticus could be the result of antibiotic pressure. The isolation of S. epidermidis from blood culture should be correlated well clinically and preferably from paired samples. There is a need for speciation of CoNS and proper study of their antibiogram for more effective patient care. Clinicians may take cognizance of the recommendation for local susceptibility patterns to be reviewed periodically when choosing the appropriate antibiotic in managing their patients.
Conflicts of interest
The authors have none to declare.
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