|Year : 2004 | Volume
| Issue : 2 | Page : 97-103
In vitro susceptibility pattern of acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides
K Prashanth , S Badrinath
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry - 605 006, India
Presently at Laboratory of Molecular and Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nacharam, Hyderabad - 500 076, India
PURPOSE: Acinetobacter spp. is an emerging important nosocomial pathogen. Clinical isolates of this genus are often resistant to many antibiotics. The in vitro susceptibility of Acinetobacter isolates obtained from patients were tested for currently used antibiotics. In addition, the study aimed at biotyping of Acinetobacter baumannii. METHODS: A total of 66 isolates were phenotypically characterised through a large panel of 25 carbon assimilation tests and susceptibility through disc diffusion method with 10 antimicrobial agents were tested. MICs were determined only for second line broad-spectrum drugs such as cefotaxime, ceftazidime, amikacin, ciprofloxacin, and ofloxacin using NCCLS guidelines. RESULTS: Multiple drug resistance (MDR) was only witnessed in A. baumannii and not in other Acinetobacter species. Aminoglycosides such as amikacin, netilmicin were most active against the MDR isolates tested (60% susceptibility). Ceftazidime was more active than cefotaxime. MDR A. baumannii strains were susceptible only to amikacin, netilmicin and ceftadizime. Ciprofloxacin had poor activity irrespective of isolates belonging to different DNA groups tested (58% resistance overall, 79% among A. baumannii). Strains of Biotypes 6 and 19 of A. baumannii showed broader resistance than those of biotype 10 and others. CONCLUSIONS: Strains of A. baumannii from patients in our hospital, were generally more resistant to quinolones, -lactam antibiotics, first and second generation cephalosporins and partially resistant to third generation cephalosporins and aminoglycosides. The strains belonging to other DNA groups of Acinetobacter were comparatively less resistant than A.baumannii, except ciprofloxacin. This study suggests that, a combination therapy, using a third generation cephalosporin and amikacin, would be best choice for treating Acinetobacter infections.
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Prashanth K, Badrinath S. In vitro susceptibility pattern of acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides. Indian J Med Microbiol 2004;22:97-103
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Prashanth K, Badrinath S. In vitro susceptibility pattern of acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides. Indian J Med Microbiol [serial online] 2004 [cited 2019 Dec 16];22:97-103. Available from: http://www.ijmm.org/text.asp?2004/22/2/97/8080
The number of nosocomial infections caused by Acinetobacter spp. has increased in recent years. These gram negative bacilli are ubiquitous in nature and are highly resistant to various antimicrobial agents., Acinetobacter baumannii s the species most often associated with nosocomial infections. A. baumannii is capable of causing life-threatening infections including pneumonia, bacteraemia, and meningitis,, A.haemolyticus and is commonly associated with infections in ICU set up. Clinical manifestations may range from colonization of the skin and mucous membrane to serious disease with substantial morbidity and mortality. According to recent taxonomy, there are 21 DNA groups identified by DNA-DNA hybridization methods. Phenotypic identification of the genus Acinetobacter to the species or DNA group level poses considerable problems. This is due to significant phenotypic overlap between the strains that were genotypically closely related DNA groups such as DNA groups 1, 2, 3 and 13TU (Tjernberg and Ursing). Therefore, the name Acineobacter calcoaceticus - Acinetoabcter baumannii complex (Acb-complex) was proposed. Biotyping of Acb-complex is useful according to earlier studies.,, Since A. baumannii is known for its multidrug resistance, constant monitoring of antimicrobial susceptibility for different antibiotics will be helpful in empirical treatment of infections with these resistant bacteria.
The purpose of this study was to determine the in vitro susceptibility of Acinetobacter isolates obtained from patients in JIPMER hospital to various antimicrobial agents by disc diffusion method and agar dilution method. In addition, isolates that were difficult to identify through routine phenotypic tests, such as isolates of Acb-complex, were subjected to additional biotyping using 5 more carbon assimilation tests that were able to describe different biotypes of Acb complex. As per the recommendations of many earlier workers which states that “every future phenotypic identification system adopted should be tested against genotypically well characterised strains, so that it adds to the validity of the proposed phenotypic tests”, we have included 24 genotypically characterised reference strains to validate our phenotypic tests.
| ~ Materials and Methods|| |
Forty-nine Acinetobacter isolates obtained from blood, CSF, sputum, endotracheal aspirate and other body fluids from patients of respiratory and paediatric ICU, and other medical wards for over a period of 21 months were identified by phenotypic test schemes of Gerner-Smidt et al and Bouvet and Grimont using a panel of 25 carbon assimilation tests. These phenotypic tests were able to identify most of the genomic species except isolates belonging to DNA group 2 and 13. Seventeen isolates derived from colonisation in hospital environment and from skin sampling of patients were also investigated. Twenty-four reference strains belonging to different DNA groups that were generously provided by Dr. Gerner-Smidt (Statens Serum Institut, Denmark) and Dr. Bouvet (Institut Pasteur, Paris) were also included to validate phenotyping. The isolates belonging to Acb-complex were further biotyped using additional 5 carbon assimilation tests of carbon sources i.e., phenylalanine, levulinate, Citraconate, 4- hydroxybenzoate and L-tartarate [Table - 1]. Sixty-six strains excluding reference strains so identified by these methods were then subjected to susceptibility testing.
Antimicrobial susceptibility testing
Disk diffusion susceptibility testing was performed on Mueller Hinton agar for the following 10 antimicrobial agents (Hi-Media, Mumbai, India) with their concentration given in parenthesis: ampicillin (10mg), amikacin (30mg), cefazolin (30mg), cefotaxime (30mg), ceftazidime (30mg), ciprofloxacin (5mg), gentamicin (10mg), norfloxacin (10mg), netilmicin (30mg), piperacillin (100mg). Minimum inhibitory concentrations (MICs) were determined by agar dilution method according to the methods established by NCCLS. MICs of the isolates were checked only for third generation cephalosporins (ceftazidime, cefotaxime), aminoglycosides with higher activity (amikacin) and quinolones (ciprofloxacin, ofloxacin). All antibiotics were obtained from Hi-Media (Mumbai, India) except for ciprofloxacin, which was procured from Oxoid (Basingstoke, UK). Escherichia More Details coli ATCC 25922, and Pseudomonas aeroginosa ATCC 27853 were used as quality control organisms. The results were analyzed using WHONET software and interpreted according to NCCLS criteria.,
A total of 53 isolates were identified accurately as all the test results were comparable with respective standard reference strains belonging to different species. Remaining 13 isolates that gave erroneous results with two or three tests differing from the reference DNA group strains were repeatedly subjected to assimilation tests to rule out contamination. Among these, six strains could be identified, wherein they gave test results similar to one of the strains of different DNA group after repeat testing. Three more isolates belonged to either DNA group 2 (Biotype 9) or DNA group 13 according to Gerner-Smidt. Remaining four isolates could not be assigned to any DNA group as their test results were highly variable from that of reference DNA groups. Hence, only 89% (53+6 =59) of them were identified correctly among all the isolates and 11% of isolates (4+3 =7) were not identifiable.
According to the phenotypic identification system followed by Gerner-Smidt, our collection had 42 strains belonging to Acb-complex. Thirty-two out of these 42 strains were correctly identified as A. baumannii (DNA group 2). Three other strains from Acb-complex identified as biotype 9 belonged to either DNA group 2 or DNA group 13. Four strains include genomic species 3 and DNA group13 (two each). Remaining 3 isolates clearly belonged to Acb-complex but were unable to assign themselves in to any one of the DNA groups of the Acb- complex. The other strains identified and tested were A. lwoffii(12), A. johnsonii (1), A. haemolyticus (2), and A. junii (1). One strain, producing haemolysis, belonged to DNA group 14. Among a total of 42 Acb-complex strains that were subjected to biotyping, biotype 10 was the most common type found in the present study (10 strains), followed by biotype 6, which comprised of 7 strains. Six strains belonged to biotype 9. Biotype 16 and 11 constituted 4 and 3 strains respectively. Biotype 2, 3, 7, 8, 15 and 19 comprised of two strains each.
The disc diffusion susceptibility testing results are given in [Table - 2], which shows the percentages of resistance, susceptibility and intermediate range among all isolates.
High levels of resistance were recorded for ampicillin (85%), cefazolin (92.2), gentamicin (63%), piperacillin (88%) and norfloxacin (69%). Netilmicin and amikacin showed maximum activity with an overall low level resistance of 32.8 and 34.4% respectively. Strains of Acb-complex were found to be more resistant to all the antibiotics tested as compared to A. lwoffii, A. haemolyticus, A. junii and strains of other DNA groups. Intermediate levels of susceptibility were encountered more in cefotaxime (37.5%) and ceftazidime (30%). Quinolones, piperacillin, gentamicin and amikacin showed highest activity against the non-A. baumannii strains. Only netilmicin and amikacin were effective against A. baumannii.
[Table - 3] shows the range of the MIC results obtained as well as the MIC90, MIC50 and percentage of resistant isolates encountered among the 66 isolates, for five second line antibiotics.
High frequencies of resistant isolates were found against ciprofloxacin and cefotaxime (58 and 42% of resistant isolates respectively). The susceptibility to amikacin and ceftazidime was 60 and 58% respectively. However, 17 strains of A. baumannii showed high level resistance to amikacin (>128µg/mL) and only six strains showed MIC>64µg/mL for ceftazidime. The MIC50 and MIC90 of amikacin were 2 and ³256µg/mL respectively. Among the cephalosporins, ceftazidime was superior antibiotic than cefotaxime as their MIC50 was 4 and 32 µg/mL respectively. The MIC50 and MIC90 for ciprofloxacin were 8 and 256 µg/mL respectively. In [Table - 4], the MICs for the 32 strains of A. baumannii, is summarized.
Resistance patterns among nosocomial bacterial pathogens may vary widely from country to country at any given point and within the same country over time. Because of these variations, a surveillance of nosocomial pathogens for resistograms is needed for every country in order to guide appropriate selection for empiric therapy. Resistance monitoring could also be a primary pointer for the emergence of an outbreak. Detection of resistance in a particular pattern may suggest a currently occurring epidemic in the hospital. However, antibiograms alone may not be sufficient to distinguish two strains that were responsible for outbreaks.
MDR A. baumannii has been implicated in several nosocomial outbreaks more often than other Acinetobacter species.,, The incidence of nosocomial infections by Acinetobacter is less reported in India when compared to other south Asian countries., Only few reports from India have used new molecular classification for describing various species of genus Acinetobacter.,, Antibiotic resistance is a major problem for patients infected with all Acinetobacter species, especially those with A. baumannii.,, This affects the selection of appropriate antibiotics for treating such patients. Only few authentic data are available regarding in vitro susceptibility of clinical isolates of A.baumannii in India., The first line therapy for Acinetobacter infections includes amikacin, imipenem, ceftazidime, or a quinolone. Imipenem monotherapy have also been proved effective in many studies. However, many recent studies have reported increasing resistance to imipenem. Most of the recent reports recommend combination therapy in the present context to avoid further resistance development to imipenem, the antibiotic once regarded as drug of choice for Acinetobacter infections., In the present study, we did not include imipenem antibiotic for susceptibility testing of our isolates for three reasons : firstly, the non-availability of the drug in the pure form; secondly, the drug has not been formally introduced in India; and thirdly, this antibiotic is not used for therapy in our hospital. Clinically significant isolates of Acinetobacter spp. other than A. baumannii, when tested, showed susceptibility to most of the antibiotics in our study.
By the disc diffusion method it was clear that aminoglycosides were still active against more than half of our isolates as they showed least resistance to amikacin and netilmicin (34 and 33% resistance-respectively). Increasing resistance for cephalosporins was observed mainly in strains belonging to Acb-complex. All other antibiotics tested showed resistance ranging from 68.8 to 92.2% suggesting that most of the first generation drugs were ineffective. Thus, agents which were used in 1980s to treat Acinetobacter infections were now inactive against this bacterium and consequently these antibiotics are not useful in treating Acinetobacter infections.,, Amikacin and netilmicin showed maximum level of activity with susceptibility of 55 and 60% respectively. Netilmicin was superior to amikacin among aminoglycosides, perhaps because this is least used in our hospital. Intermediate levels of susceptibility were encountered more in cefotaxime (37.5%) and ceftazidime (30%). These results were very similar with that of earlier multicentric study. In our study, amikacin and ceftazidime were still active against most of the Acinetobacter strains irrespective of species they belonged. However, we noted that the MIC ranges of our strains were higher than those found in the study of Chang et al. Strains of Acb-complex were more resistant to all the antibiotics tested as compared to A.lwoffii , A.haemolyticus , A.junii and strains of other DNA groups. Piperacillin, gentamicin and amikacin showed the highest activity against non-A.baumannii species. These results are in concordance with earlier reports.,, Amikacin was comparatively effective against all Acinetobacter isolates (60% susceptibility). However, 50% of our A. baumannii strains were resistant to amikacin with 17 strains of A. baumannii showing high level resistance to amikacin (>128µg/mL). In contrast, Chang et al reported higher susceptibility rates (74.5%) among A. baumannii strains for amikacin. Amikacin was introduced in our hospital only in 1997. This moderate resistance development within a short period might be due to extensive use of this useful drug in our ICUs. This observation is supported by a recent investigation conducted in New York, wherein a reversal trend in resistance for amikacin has been attributed to decline in its use.
Less resistance levels were also noticed for ceftazidime (37%). This is different from the results reported from Turkey and Greece, wherein 67.5 and 96% resistance was witnessed respectively for ceftazidime., The proportion of strains susceptible to ceftazidime irrespective of different species of Acinetobacter was only 58% at £ 32mg/mL, which is slightly lesser than earlier reports by Visalli et al (71%) for strains collected from parts of the US, Switzerland, Netherlands and Jones et al (63%) for strains collected from 50 US medical centers. However, both ceftazidime and cefotaxime had more strains showing intermediate range of susceptibility (20%). This relatively high frequency of A.baumannii strains with moderate resistance to cephalosporins in fact anticipates future problems with increase in resistant development as a consequence of uninterrupted and indiscriminate use of these antimicrobials. Interestingly, irrespective of species or different DNA groups of Acinetobacter tested, all our isolates showed higher resistance to ciprofloxacin (58% resistance overall; 79% among A.baumannii). This was in concordance with one muticentric study. High percentages of strains belonging to A. baumannii were resistant to ciprofloxacin, ofloxacin and cefotaxime (79, 76 and 54% respectively) by agar dilution method. A.baumannii strains were more resistant to quinolones when compared with other studies in Chile., Chang et al reported highest activity of quinolones against A. baumannii having susceptibility percentage of 97.8%. In contrast, quinolones had least susceptibility of 21% in our study, very similar to the results seen in Germany., This high level of resistance to ciprofloxacin may be explained by the fact that ciprofloxacin was introduced in the late 1970s in our region against Salmonella More Details typhi and since that time this antibacterial agent has been extensively used in our hospital. However, among quinolones, ofloxacin was slightly more active than ciprofloxacin against Acinetobacter species in our study as there was one fold decrease in MIC for ofloxacin as against ciprofloxacin.
Most published studies consider A.baumanni isolates as a homogeneous taxonomic group, even though 19 biotypes within A.baumanni have been recognized. Resistance patterns of different biotypes may help in initial tracing of an epidemic. In our study, we found biotype 10 and biotype 6 being most prevalent in this geographical area. Biotype 10 has been not encountered frequently in other studies. Biotypes 6 and 19 showed high frequency of resistance in our study, which is in contrast with the study from Germany wherein strains of biotype 9 of A. baumannii were more resistant than biotype 6. In fact, in one study more than 70% of biotype 9 and 8 strains were resistant to 3rd generation cephalosporin, whereas most strains of biotype 6 were susceptible. Almost all strains of biotype 6 were resistant to amikacin and ciprofloxacin in our study where as, Bello et al had higher number of susceptible biotype 6 strains for amikacin and ciprofloxacin in their collection, accounting for 85 and 80% susceptibility respectively. In our study, comparatively high resistance was observed among biotype 10 strains (50%). All other biotypes were susceptible to most of antibiotics and resistant to ciprofloxacin and ofloxacin. However, Bello et al reported that 'other biotypes' (biotypes except 6 and 10) were more susceptible to ciprofloxacin. Since prevalent biotypes of A. baumannii differ according to the countries, additional and more exhaustive comparative information dealing with antimicrobial susceptibility and other properties of isolates of different biotypes of A. baumannii is needed.
In summary, strains of A. baumannii from patients in our hospital were generally more resistant to quinolones, b-lactam antibiotics, first and second generation cephalosporins and partially resistant to third generation cephalosporins and aminoglycosides. The strains belonging to other DNA groups of genus Acinetobacter were comparatively less resistant than A. baumannii, except ciprofloxacin. However, despite such resistance, combination therapy, using a third generation cephalosporin and amikacin, could be the best choice for treating Acinetobacter infections in our setup.
We thank Dr. P Gerner-Smidt (Statens Serum Institut, Denmark) and Dr. Philippe JM Bouvet (Institut Pasteur, Paris) for generously providing us Acinetobacter reference strains of different DNA groups.
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