Mar 29, 2012 - L. acidophilus, 2 L. fermentum), Micrococcus (1. M. agilis), Pragia (1 P. fontium), Proteus (3 P. mirabilis, 1 P. penneri, 3 P. vulgaris), Pasteurella.
Noto-are 14687587: Medicine. 2012-03-29.
Effect of Aerobic and Microaerobic Growth Conditions on Antimicrobial Sensitivity of Important Bacterial Isolates from Clinical Samples and on Minimum Inhibitory Concentration of Gentamicin, Vancomycin, Ciprofloxacin and Tetracycline Bhoj Raj Singh Indian Veterinary Research Institute Izatnagar Bareilly Uttar Pradesh INDIA
Abstract A total of 174 microbial strains including one Candida albicans and 173 commonly occurring bacterial strains belonging to 20 genera were tested against disks of 21 commonly used antimicrobials to evaluate effect of aerobic and microaerobic growth conditions on sensitivity. Minimum inhibitory concentration (MIC) of vancomycin, gentamicin, tetracycline and ciprofloxacin was determined against 20 strains for each drug under aerobic and microaerobic growth. Zone of inhibition against antimicrobial disks was significantly narrower (paired T-test, p, 0.05) under microaerobic condition for erythromycin, amoxcillin+clavulanic acid, ciprofloxacin, clindamycin, co-trimoxazole, norfloxacin, gentamicin, streptomycin, azythromycin and cefaperazone+sulbactam
than under aerobic conditions. However, zone of inhibition was significantly (p, 0.05) larger around tetracycline disks under microaerobic environment than under aerobic growth conditions. Decision to sensitivity of bacteria was found significantly altered for ciprofloxacin, clindamycin, co-trimoxazole, nitrofurantoin, gentamicin and azythromycin under paired T test (p, 0.05). However, using Chi-squired test0.05 test the difference was significant only for azythromycin, gentamicin, clindamycin and norfloxacin under aerobic and microaerobic growth conditions. The difference was significant (Chi-squire test, 0.05) for enterococci against clindamycin and norfloxacin (specifically E. avium), for Escherichia coli against gentamicin and azythromycin, and for Klebsiella against nitrofurantoin and streptomycin. MIC tests were significantly different (paired T-
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test0.05, F-test0.05, Chi-squire test 0.05) for gentamicin and vancomycin while difference was quite significant for ciprofloxacin (MIC increased under microaerobic conditions) and tetracycline (MIC decreased under microaerobic conditions). The study suggested that there is a need for change in antimicrobial drug sensitivity testing procedure particularly for facultative anaerobes. Keywords: Bacteria, antimicrobial sensitivity, MIC, aerobic, microaerobic
The study suggested that in antimicrobial activity of a drug observed in standard antimicrobial drug sensitivity assay may be misleading and clinician may not get the predicted results for a drug because the slight change in oxygen concentration significantly modulate the antibiotic sensitivity and MIC of the commonly used antimicro-
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bials. This study indicated a need to reevaluate the methodology of antibiotic sensitivity testing particularly for facultative anaerobes, which are major class of pathogenic bacteria. 1. Introduction Since long the pros and cons of in vitro antimicrobial drug sensitivity of pathogenic bacteria remained a matter of hot discussions and speculations. At several occasions it is felt that laboratory tests for antimicrobial drug sensitivity fail to recommend the curative drug. In 1989, it was observed that extra oxygen in sensitivity testing environment (hyperoxia) significantly enhanced bacteriostatic activity of nitrofurantoin and trimethoprim and decreased their MIC [1]. Hyperoxia enhanced bactericidal action of trimethoprim i.e, decreased MBC for E. coli and P. aeruginosa. However, hyperoxia did not affect MICs of gentamicin or tobramycin. Thus it was concluded that hyperoxia potentiates the antimicrobial activity of the reduction-oxidationcycling antibiotics [1]. A study on E. coli concluded that reactive oxygen has important role in antibacterial action of ciprofloxacin and antioxidant as glutathione may render protection against fluoroquinolones [2]. Similarly, studies on Pseudomonas aeruginosa revealed that presence of oxygen enhances the antimicrobial activity of tobramycin and ciprofloxacin even in biofilms, i.e., microaerophilic and anaerobic conditions favored survival of bacteria even in presence of bactericidal drugs. It was evident through transmission electron microscopic observations of antibiotic-affected cells, they were lysed, vacuolated, and elongated exclusively near the air interface in antibiotic-treated biofilms while cells un-
derneath beyond reach of oxygen were healthy in presence of antibiotics [3, 4]. Growth under anaerobic conditions not reduced the ability of ceftazidime, meropenem, piperacillin, livofloxacin, or piperacillin/tazobactam to inhibit planktonic growth of P. aeruginosa and MIC50 values for these antibiotics remain the same as under aerobic conditions. However, tobramycin, amikacin, and aztreonam have less affectivity to inhibit growth under anaerobic conditions, and their MIC50 increases two to four fold and bactericidal activity decreases about fourfold [5, 6]. To clear majority of confusions CLSI [7, 8] played a crucial role through setting the standards of testing and concluding the results for antimicrobial sensitivity for different pathogens. Further, some studies on microaerophilic [9] and aerobic bacteria5, 6 have shown the effect of anaerobic growth conditions on their sensitivity to different antimicrobials but little is explored on a major group of pathogens, facultative anaerobes, that too under restricted oxygen levels increased carbon-di-oxide tension. Therefore, it is still worth experimenting to understand effect of microaerobic conditions on microbial drug sensitivity because our body conditions are largely microaerobic and testing of facultative anaerobes under recommended aerobic conditions may not be of true value. Thus, this study of comparison of antimicrobial drug sensitivity under aerobic (standard) and microaerobic growth conditions was conducted on several clinical and reference strains of bacteria so that we can evaluate our routine methods of antimicrobial drug sensitivity.
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2. Materials and Methods Microbial strains A total of 173 microbial strains including one Candida albicans and 172 bacterial strains belonging to 20 genera viz., Bacillus (4), Bordetella (1 B. bronchiseptica), Burkholderia (1 B. cepaia, 1 B. gladiolii, 2 B. pseudomallei), Enterococcus (10 E. avium, 18 E. caecorum, 3 E. casseliflavus, 2 E. dispar, 5 E. faecalis, 1 E. facium, 5 E. gallinarum, 2 E. mundatii, 1 E. solitarius, 1 Enterococcus spp.), Enterobacter (2 E. aerogenes, 4 E. agglomerans, 1 E. amnigenus, 1 E. gregoviae), Erwinia (3), Escherichia (21 E. coli, 1 E. coli inactive, 2 E. fergusonii), Klebsiella (3 K. oxytoca, 6 K. pneumoniae), Lactobacillus (2 L. acidophilus, 2 L. fermentum), Micrococcus (1 M. agilis), Pragia (1 P. fontium), Proteus (3 P. mirabilis, 1 P. penneri, 3 P. vulgaris), Pasteurella (4 P. multocida), Pseudomonas (4), Salmonella (3 S. enterica ssp. enterica), Serratia (1, S. rubidiae), Staphylococcus (1 S. aureus, 1 S. felis), Streptococcus (1 S. equi, 21 S. mobilis, 1 S. porcinus, 7 S. pyogenes, 12 Streptococcus spp.), Xenorhabdus (1 X. bovis) and Yersinia (1 Y. enterocolitica) isolated from clinical samples were used to evaluate effect of antimicrobial activity of different antimicrobial drugs under aerobic and microaerobic growth conditions. Bacterial strains isolated from different clinical samples and available at General Bacteriology laboratory of the Institute were revived through growing in tryptic soy broth (TSB, Hi-Media Mumbai), rechecked for purity on 5% sheep blood agar (BA, Hi-Media) and identity [10, 11]. Revived cultures were maintained on nutrient agar (Hi-Media) slants during the period of the study. Besides, a Serratia rubidiae (E2) strains sensitive to all common antimicrobial drugs used as reference strains were procured from
Effect of Aerobic and Microaerobic Growth Conditions on Antimicrobial Sensitivity of Important Bacterial Isolates from Clinical Samples and on M
Microbiology Laboratory of ICAR Research Complex for NEH Region, Nagaland Centre, Jharnapani, Nagaland. Antimicrobial sensitivity assay Antimicrobial sensitivity was determined using disk diffusion assay [7, 8] against ampicillin (30 mcg), amoxycillin (30 mcg), amoxycillin+clavulanic acid (10 mcg), azithromycin (15 mcg), ceftazidime (30 mcg), cephalexin (30 mcg), cefoperazone-sulbactam (75+30 mcg), chloramphenicol (30 mcg), ciprofloxacin (10 mcg), clindamycin (2 mcg), cloxacillin (5 mcg), colistin (25 mcg), cotrimoxazole (25 mcg), erythromycin (15 mcg), gentamicin (10 mcg), nalidixic acid (30 mcg), nitrofurantoin (300 mcg), norfloxacin (10 mcg), streptomycin (25 mcg), tetracycline (30 mcg), vancomycin (30 mcg) disks under aerobic as well as microaerobic growth conditions at 37oC and zone of inhibition was measured in millimeters (mm) and results were concluded as sensitive or resistant as per CLSI [7, 8]. All the tests were repeated and unless two matching of zone of inhibition were recorded for an antimicrobial drug for the individual bacterial strain. Determination of minimum inhibitory concentration (MIC) To determine minimum inhibitory concentration under aerobic and microaerobic conditions of growth, 32 strains of different bacteria (20 against each antibiotic) were tested against vancomycin, gentamicin, tetracycline and ciprofloxacin HiComb strips (Hi-Media) on Mueller Hinton agar (HiMedia) plates as described by the manufacturer [8]. The tests were conducted in triplicate and values recorded common at least in two tests were recorded as MIC values. Generation of microaerobic growth conditions Anaerocult (1.16275.0001, Merck, Germany) was used as per recommendation of the manufacturer for generation of mi-
croaerobic medium in sealed microaerobic jar. Incubation was made at 37oC for 24-48 h before reading the results for all bacteria under test. Statistical analysis Sensitivity data was analyzed for evaluating the effect of growth conditions on zone of growth inhibition around antimicrobial disks and MIC values using paired T-test and F test while to compare the processed data (results classified as sensitive or resistant) Chi-square test (CT) was used in Microsoft Exel. All the test results were evaluated at 5% level of significance (p, 0.05). 3. Results An appreciable difference able to change the conclusion for sensitivity was evident for different bacteria under aerobic and microaerobic conditions. Different bacteria varied for resistance under aerobic/ microaerobic conditions to ampicilin (64.2%/ 67.2%), amoxycillin (32%/ 28%), amoxycillin+clavulanic acid (27.7%/ 33.8%), azythromycin (10.5%/ 52.6%), ceftazidime (45.5%/ 36.4%), cephalexin (21.5%/ 23.1%), cefoperazone-sulbactam (0%/ 5.3%), chloramphenicol (17.8%/ 24.4%), ciprofloxacin (9.8%/ 12.1%), clindamycin (29.2%/ 49.2%), cloxacillin (33.8%/ 33.8%)), colistin (41.5%/ 43.9%), cotrimoxazole (47.3%/ 55%), erythromycin (34.2%/ 38.2%), gentamicin (4.8%/ 15.2%), nalidixic acid (28.6%/ 32.1%), nitrofurantoin (37.8%/ 45.6%), norfloxacin (27.3%/ 37.8%), streptomycin (29.6%/ 35.2%), tetracycline (21.8%/ 19%) and vancomycin (72.2%/ 70.4%). Paired T-test for comparison of zone of inhibition against antimicrobial disks revealed that it was significantly (p, 0.05) narrower under micro-aerobic condition for erythromycin, 3
amoxcillin+clavulanic acid, ciprofloxacin, clindamycin, co-trimoxazole, norfloxacin, gentamicin, streptomycin, azythromycin and cefaperazone+sulbactam than under aerobic conditions. However, zone of inhibition was significantly (p, 0.05) larger around tetracycline disks when tested in microaerobic environment than observed under aerobic growth conditions. Although effect of several antimicrobials appeared to be severely affected while reading the zone of inhibition, final results to decision of sensitivity of bacteria was found significantly (p, 0.05) altered only for ciprofloxacin, clindamycin, co-trimoxazole, nitrofurantoin, gentamicin and azithromycin under paired T test. However, on putting the test results for Chi-squire test0.05 (CT0.05), difference was significant only for azythromycin, gentamicin, clindamycin and norfloxacin, more number of strains were sensitive under under aerobic than under microaerobic growth conditions. On further evaluation of difference in drug sensitivity of different bacteria under aerobic and microaerobic conditions of growth, significantly (CT0.05) more strains of enterococci were sensitive against clindamycin and norfloxacin (specifically for E. avium) under aerobic conditions than in microaerobic growth. Similarly more strains of Escherichia coli were sensitive to gentamicin and azythromycin, and more number of Klebsiella was sensitive to nitrofurantoin and streptomycin under aerobic environment than under microaerobic. The difference in sensitivity under aerobic and microaerobic conditions was appreciable for several bacterial strains though statistically insignificant (p more than 0.05) for other bacteria under test. On comparison of MIC determined through E-test (20 different strains of bacteria for
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each antibiotic, Table 1) no significant (p more than 0.05) difference was appreciable either using paired T-test0.05 or F-test0.05 or CT0.05 test for gentamicin and vancomycin while difference was quite significant for ciprofloxacin (MIC increased under microaerobic conditions) and tetracycline ( MIC decreased under microaerobic conditions).
Fig. 1: Minimum inhibitory concentration in micrograms of antibiotics under aerobic and microaerobic growth conditions against common bacteria
4. Discussion It is understood that the in vitro tests for most of the biological effects may predict reliable outcome but it may not be the same as observed in vivo and fails to predict the real outcome in biological system. It is specifically true for many of
the antimicrobials viz., the in vitro resistance of H. pylori to metronidazole but in vivo the drug cured the infection [9]. This discrepancy is supposed to be due to the resistance observed in culture under microaerophilic conditions and growth of the bacterium in its ecological niche facing a certain period of time under temporarily anaerobic conditions which might be causing the reduction of metronidazole in previously resistant strains with subsequent killing of the bacteria [9]. In the present investigation most of the antimicrobials (erythromycin, amoxcillin+clavulanic acid, ciprofloxacin, clindamycin, co-trimoxazole, nitrofurantoin, norfloxacin, gentamicin, streptomycin, azythromycin and cefaperazone+sulbactam) were found more effective against most of the facultatively anaerobic bacterial strains under recommended aerobic conditions of testing than under microaerobic environment expected under infective stage of their growth. Although there is no earlier comparable observation, antimicrobial effect of nitrofurantoin, trimethoprim and fluoroquinolone has been reported better in hyperoxic condition than under aerobic standard conditions of growth [1, 2]. It indicate that oxygen actively modulate the antibacterial effect of antimicrobials, i.e., decreasing the MIC with increase in oxygen. However, observations appear to contrast earlier observations pertaining to no effect of hyperoxia on antimicrobial effect of aminoglycosides [1]. In this study zone of inhibition was significantly decreased around disks of both gentamicin and streptomycin under microaerobic conditions. This discrepancy may be due to alteration in drug metabolism under microaerobic conditions while earlier observations were made in aerobic and hyperoxic environment only [1]. Although there was 4
an apparent effect on sensitivity pattern of bacteria against gentamicin, studies on MIC of gentamicin indicated insignificant variation under microaerobic and aerobic environment, similar to earlier observations [1] under aerobic and hyperoxic conditions of growth. Results on effect of aminoglycosides appear to contrast previous observations of fourfold increase in MIC of amikacin, an aminoglycoside, against P. aeruginosa under anaerobic conditions [5, 6]. However, the contrast may not be true because of in this study too if we take only a few strains of a specific bacteria as in earlier studies [5, 6] MIC increased by 5 fold for a Streptococcus spp. and 2.5 fold for a E. coli strain, thus it may be concluded that effect of oxygen on antibiotic sensitivity might be affected by bacterial strain too. Therefore, to have a general idea about the effect of oxygen on effect of any antibiotic study on more number of strains of different groups might be more ideal. Observations have revealed that effect of microaerobic condition of growth was not uniform on different bacteria as Enterococcus avium were significantly more sensitive to clindamycin and norfloxacin under aerobic conditions than other bacteria. Similarly, Escherichia coli were more sensitive to gentamicin and azythromycin, and Klebsiella to nitrofurantoin and streptomycin under aerobic environment than other facultatively anaerobic bacteria. The difference in sensitivity of different bacteria under aerobic and microaerobic conditions might be due difference in metabolism at micro level and the observations also explains the discrepancy in observations of different studies conducted on limited number of strains of specified pathogen. Although there was no significant increase in MIC of vancomycin as whole on different
Effect of Aerobic and Microaerobic Growth Conditions on Antimicrobial Sensitivity of Important Bacterial Isolates from Clinical Samples and on M
bacteria, for few strains (S. enterica) it increased significantly (Fig. 1) under microaerobic conditions of growth. In contrast, increase in MIC of ciprofloxacin under microaerobic condition of growth was significant on most of the bacteria. The observation on increased MIC of ciprofloxacin under microaerobic environment appears to be in concurrence to earlier observations [2, 5] predicting the same under reducing environment. Although most of the antimicrobial drugs significantly affected either for their effect on bacteria or MIC, microaerobic environment had adverse effect, effect on tetracycline affectivity was mostly positive and its MIC was significantly lower for most of the bacteria than in aerobic environment. The study indicated that a pathogen in standard method of sensitivity assay might be resistant to tetracycline in laboratory while the drug may be useful on use in clinical practice. It might be the indirect reason behind the popularity of the tetracyclines among veterinary practitioners in India [12] despite of reported widespread tetracycline resistance in common pathogenic bacteria [13]. The variation in resistance to several antimicrobial drugs under microaerobic conditions might be due to modulation of expression of different genes under aerobic, anaerobic and microaerophilic conditions, including several genes associated with antimicrobial susceptibility of many bacteria [14]. It is observed earlier that under suboptimal growth conditions in biofilms E. coli become less sensitive to antimicrobial action of monochloramine [15], while Streptococcus mutans may become more resistant to many of the antimicrobials [16]. However, it cannot be generalized all strains of a species of bacteria as observed in the present study (Fig. 1) because different strains of bacteria may not have similar ability
to handle stress as reported in E. coli strains due to their different abilities to cope with ROS [17]. 1. Conventional antimicrobial drug testing in microbiological laboratory under aerobic incubation may be misleading and risky when facultative anaerobes are causing infection. References [1] Muhvich K H, Park M K, Myers R A M &Marzella L, “Hyperoxia and the antimicrobial susceptibility of Escherichia coli and Pseudomonas aeruginosa”, , Vol. 33, No. , 1526-1530, 1989. [2] Goswami M, Mangoli S H &Jawali N, “Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli”, , Vol. 50, No. , 949954, 2006. [3] Walters III M C, Roe F, Bugnicourt A, Franklin M J &Stewart P S, “Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin”, , Vol. 47, No. , 317-323, 2003. [4] Borriello G, Werner E, Roe F, Kim A M, Ehrlich G D &Stewart P S, “Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms”, , Vol. 48, No. , 26592664, 2004. [5] Field T R, White A, Elborn J S &Tunney M M, “Effect of oxygen limitation on the in vitro antimicrobial susceptibility of clinical isolates of Pseudomonas aeruginosa grown planktonically and as biofilms”, , Vol. 24, No. , 677-687, 2005. [6] King P, Citron D M, Griffith D C, Lomovskaya O &Dudley M N, “Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients”, , Vol. 66, No. , 181-186, 2010. 5
[7] , “Performance standards for antimicrobial susceptibility testing, 18th informational supplement, CLSI document M100-S18”, , Vol. , No. , , 2008. [8] , “Method for dilution antimicrobial susceptibility tests for bacterial that grow aerobically; approved standard. 8th edn, CLSI document M07-A8”, , Vol. , No. , , 2009. [9] van Zwet A A, Thijs J C &de Graaf B, “Explanations for High Rates of Eradication with Triple Therapy Using Metronidazole in Patients Harboring Metronidazole-Resistant Helicobacter pylori Strains”, , Vol. 39, No. , 250-252, 1995. [10] Holt J G, Sneath P H A, Mair M S &Sharpee M E, “Bergey,s manual of systematic bacteriology”, , Vol. 2, No. , , 1986. [11] Singh BR, “Labtop for Microbiology Laboratory”, , Vol. , No. , , 2009. [12] Singh BR, “Thermotolerance and multidrug resistance in bacteria isolated from equids and their environment: source of pasteurization resistant bacteria.”, , Vol. 164, No. , 746-750, 2009. [13] Singh BR, “Antimicrobial drug uses by veterinarians in equine clinical cases in India”, , Vol. 3, No. , 165-178, 2010. [14] dos Santos Nobre L I, “Unravelling novel modes of antimicrobial action”, , Vol. , No. , 188, 2010. [15] Berry D, Xi C &Raskin L, “Effect of Growth Conditions on Inactivation of Escherichia coli with Monochloramine”, , Vol. 43, No. , 884-889, 2009. [16] Xue X, Sztajer H, Buddruhs N, Petersen J, Rohde M, Talay S R &Wagner-Dobler I, “Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans.”, , Vol. 6, No. , , 2011. [17] Semchyshyn H, Lushchak V &Storey K, “Possible reasons for difference in sensitivity to oxygen of two Escherichia coli strains”, , Vol. 70, No. , 4424-4431, 2005.
