The resistance patterns of 100 local strains of Ps. aeruginosa were investigated using two different methods: (1) Broth Dilution and (2) ε-test. From the seven antibiotics tested, Ps. aeruginosa showed a 100% sensitivity to imipenem (n=30). Among the first-line agents, azlocillin, ceftazidime, and gentamicin showed the highest sensitivity rates, 87%, 93% and 92% respectively. Aztreonam and ciprofloxacin showed the presence of intermediately sensitive strains, with 61% of the isolates tested being fully sensitive to each antibiotic. Only 47% of the strains were found to be sensitive to ceftriaxone. The results obtained were similar to studies carried out abroad.peer-reviewe

Borg, Michael Angelo

Zarb, Peter

English

OAR@UM

11 t.IalLeSe Medical Journal, 1998; 10(1): 11 All rights reserved Antibiotic susceptibility patterns of local strains of Pseudomonas aeruginosa Peter Zarb ,* Michael A. Borg ** ABSTRACT: The resistance patterns of 100 local strains of Ps. aeruginosa were investigated using two different methods: (1) Broth Dilution and (2) £-test. From the seven antibiotics tested, Ps. aeruginosa showed a 100% sensitivity to imipenem (n=30). Among the first-line agents, azlocillin, ceftazidime, and gentamicin showed the highest sensitivity rates, 87%, 93% and 92%, respectively. Aztreonam and ciprofloxacin showed the presence of intermediately sensitive strains, with 61 % of the isolates tested being fully sensitive to each antibiotic. Only 47% of the strains were found to be sensitive to ceftriaxone. The results obtained were similar to studies carried out abroad. * Department of Phannacy, University of Malta. Msida, Malta. **Department of Pathology, St Luke's Hospital, Gwardamangia, Malta. Correspondence: Dr. M.A. Borg, Infection Control Unit, SI. Luke's Hospital, Gwardamangia, Malta. Keywords: antibiotic susceptibility testing, Pseudomonas aeruginosa, minimum inhibitory concentrations, minimum bactericidal concentrations, tolerance, multiple drug resistance Introduction Ps. aeruginosa can colonise healthy individuals as a harmless saprophyte. It is known to be a major cause of nosocomial infections particularly as it grows virtually everywhere including respirators used in operating theatres. It is one of the most important hospital pathogens especially in patients with a degree of immunosuppression. It is important because many strains show a degree of multiple drug resistance. The degree of resistance to several antibiotics among clinical isolates of Ps. aeruginosa has been increasing in recent years. Bronchopneumonia, septicaemia, meningItIS, and urinary tract infections caused by Ps. aeruginosa may be acute or chronic in relation to the host immune reactivity. Ps. aeruginosa is also a cause of severe epidemic diarrhoea in infants, ocular and bum infections, osteomyelitis and malignant external otitis. The pathology of a Ps. aeruginosa infection includes necrosis and haemorrhagic lesions, infiltration by mononuclear cells, cytotoxic effects on macro phages and polymorphonuclear leukocytes, and widespread granulomatous lesions. According to the National Nosocomial Infections Surveillance System (NNIS), Ps. aeruginosa is the third most important nosocomial pathogen (11 %) I. Methods The samples used in this study were taken from clinical isolates which were identified as Ps. aeruginosa by the laboratory staff as part of their routine work. (1) The Broth Dilution Technique Iso-Sensitest Broth® was used for this method. Each sample that did not show growth was inoculated on Blood Agar to determine whether a particular concentration was inhibitory or bactericidal. The lowest concentration (i.e., highest dilution) which did not show growth on the agar plate was the MBC. By determining both MIC and MBC tolerance was also estimated. Tolerance is of the utmost importance in cases where bactericidal therapy is a requirement. (2) £-Test technique Half the plate was swabbed with the control strain (ATCC 27853), the other half being inoculated with the test strain so that in using this procedure each £-test strip had a simultaneous control run2. £-Test MIC values have been shown to be reproducible and directly proportional to MIC values from the NCCLS reference agar dilution procedure3. Results The MIC50 and MIC90 data together with the MIC range for the seven antibiotics tested are shown in Table l. Figure 1 shows the qualitative classification of the data obtained. Isolates are classified as Sensitive, Intermediate or Resistant to the respective antibiotic. The breakpoints followed were those recommended by the European Committee for Clinical Laboratory Standards (ECCLS). Tolerance Tolerance is when the MIC/MBC ratio is 32 (i.e. the difference between MIC and MBC is ~ 6 two-fold dilution factors. A high frequency of tolerance was found with azlocillin, as illustrated in Figure 2. P. Zarb, M. A. Borg Table 1 - In vitro activity of antibacterial agents against Ps. aeruginosa. 12 Antimicrobial MIC50 MIC90 Mean MIC MIC range Breakpoints Percent Agent (f.1g/ml) (f.1g/ml) (f.1g/ml) (f.1g/ml) Susceptible Azlocillin 4 128 13.64 <8 - >256 S= <64 R= >128 87 Aztreonam 8 32 6.68 <2 i >64 s= <8 R= >32 81 Ceftazidime 2 8 3.03 <2 - >64 S= <16 R= >32 97 Ceftriaxone 64 64 24.06 2 - >32 s= <16 R= >32 47 Ciprofloxacin 1 16 1.04 <0.25 - >8 S= <1 R= >4 79 Gentamicin 2 8 2.69 <1 - >32 S= <4 R= >8 92 Imipenem 1 2 1.17 0.25 - 4 S= <4 R= >16 100 Fig. 1 - Qualitative Results Multiple Drug Resistance Multiple Drug Resistance (MDR) is yet another distressing problem. Figure 3 shows the resistance patterns of the 100 isolates studied. All the strains were fully sensitive to imipenem. The criterion chosen for defining Multiple Drug Resistance was that an isolate results resistant to two or more antimicrobial agents excluding ceftriaxone which is not an antipseudomonal cephalosporin. Thus a total of 14 isolates were classified as MDR. Figure 3 shows the resistance patterns of the 100 isolates studied. Comparison with other studies Table 2 summarises the comparison of results obtained cQ) Q) c E@ E c '0 '0 Q)C1l E c 	 in this study with studies carried out abroad. The results '0 c 0 C1l Q)'5 x 'E 0 0 x N 'N C1l 0 C1l a. 	 obtained in different studies follow the same trend in C1l c;::C1l ~ .t:: ~ e i: Q) :§ 	 resistance and any differences in results are mainly due Q) Q)C1l Cl() () a. '0 	 to geographical variation in resistance patterns. The only relevant difference is , with ciprofloxacin to which Maltese strains show higher levels of resistance. sensitivity Discussion ToleranceFig. 2 - Tolerance If a species has high resistance to an antibiotic (e.g., ceftriaxone, Figure 1), this can be detected by routine Fig. 3 - Multiple Drug Resistance 50 45 40 35 f/) ~ 30 (5 '" !!l 25 '0 20ci z 15 10 5 c Q) cE c '0'B c '" E '0 'E 	 0 .. ­0 0 x N ~ 0 '" .9 	 o 234 5~ c;:: cN <= '" e Q)'" 	 Clfl a.'" '0 No. of Antibiotics to which each 100 90 80 70 60 % 50 40 30 20 10 0 f/) c 25 20 .~ 15 en 'E ~ ~ 10 ~ o 5 0 Antibiotic 	 Isolate was Resistant 13 Antibiotic susceptibility patterns of local strains of pseudomonas aeruginosa Table 2. Comparison with other studies. Antibiotic 	 Sensitivity (%) Nation Author and year Malta Abroad Azlocillin 87 62 Germany Ansorg. et al. . 19904 Aztreonam 81 78.3 Multicentre study Verbist. 19945 88.0 Germany Abb.19926 90.4 Greece Sofianu. et al . 19897 Ceftazidime 97 91.7 Multicentre srudy Verbist. 19945 93.0 Germany Abb. 19926 82 Germany Ansorg. et al.. 19904 93.0 Greece Sofianu. et al, 19897 Ceftriaxone 47 <50 Germany Ansorg. et al.. 19904 62.0 Greece Sofianu. et al.. 19897 Ciprofloxacin 79 90.0 Germany Abb.19926 90 Germany Ansorg. et a1.. 19904 Gentamicin 92 84.0 GermaQY Abb.19926 38.4 Greece Sofianu. et al.. 19897 Imipenem 	 100 93.5 Germany Abb.19926 91 Germany Ansorg. et al.. 19904 99.5 Greece Sofianu. et al.. 19897 laboratory investigations. On the other hand, tolerance is only detected if actual MICs and MBCs are measured. Thus, an antibiotic such as azlocillin seems to be active against Ps. aeruginosa (Figure I), but in practice, many number of strains are actually tolerant to it (Figure 2). This is important in patients who require bactericidal drugs. Multiple Drug Resistance All three isolates which were resistant to ceftazidime showed cross resistance with ceftriaxone. However, four MDR isolates were sensitive to ceftriaxone and resistant to unrelated agents. This is important since only 47% of the isolates were sensitive to ceftriaxone. Thus, there is still a place for the use of ceftriaxone as an antipseudomonal agent even though more than 50% of isolates are resistant. Conclusions Ps. aeruginosa is one of the most important pathogens in hospitals, accounting for 11 % of all hospital acquired infections, according to the National Nosocomial Infections Surveillance System, 19891. Therefore, its resistance to a number of antimicrobial agents poses problems in the empiric selection of an appropriate drug for the treatment of infections caused by this organism. According to the results obtained, ceftazidime and gentamicin should be the empirical first line drugs to use Table 3. Health Service cost (Lm) of parenteral therapy Antibiotic Dose/phial Cost/phial Dose Regimen Cost for 7 days Azlocillin 2.0g 1.94 2.0g t.d.s. 40.74 Aztreonam 500mg 1.62 LOg t.d.s. 68.04 Ceftazidime l.Og 2.90 l.Og t.d.s. 60.90 Ceftriaxone 2.0g 10.00 2.0g b.d. 140.00 Ciprofloxacin 200mg 8.77 200mg b.d. 122.78 Gentamicin 40mg/ml 0.62 80mg t.d.s. 13.02 Imipenem 500mg 8.40 500mg t.d.s. 176.40 against Ps. aeruginosa infections since these two antibiotics are the only ones with a percentage sensitivity greater than 90. The second line agent imipenem should be reserved for strains resistant to first line agents, especially those resistant to both gentamicin and ceftazidime. When choosing the right antibiotic the price factor should be taken into account. Table 3 shows the approximate cost of treatment for a week with the seven antibiotics tested in this study. From this table one can see that gentamicin is the cheapest antibiotic whereas imipenem is the most expensive. This may be one reason why gentamicin is used as a first line agent even though it has a number of potential side effects and precautions for use, whereas, the safer antibiotic imipenem is classified as a second line agent. However, the most interesting finding was obtained by comparing resistance levels to ciprofloxacin in Malta with two studies carried out in Germany. In Malta only 79% of the isolates were sensitive to ciprofloxacin as opposed to 90% in both German studies. This suggests the overuse or misuse of ciprofloxacin in Malta. It is likely to happen with any antibiotic which can be given by the oral route. In such a case, as with ciprofloxacin, there can be overuse of the antibiotic both in hospital and especially in community practice. A study of the development of resistance by Ps. aeruginosa during therapy with ciprofloxacin was published in 19898. This is even more alarming when considering that ciprofloxacin can induce low level resistance to structurally unrelated antibiotics in Ps. aeruginosa and methicillin resistant Staphylococcus aureus9. References I. 	 Schaberg DR. Culver OH. Gaynes RP. Major trends in the microbial aetiology of nosocomial infection. Am 1 Med 1991; 91(SuppI3B): 72S-75S. 2. 	 Bignardi GE. MIC Determination by the E Test. 1. Antimicrob. Chemother 1991; 28: 773-4. 3. 	 Biodisk AB. Sweden. Etest® technical guide no.3. May 1993. 4. 	 Ansorg R.. Muller KO. Wiora 1. Comparison of inhibitory and bactericidal activity of antipseudomonal antibiotics against Ps. aeruginosa isolates from cystic fibrosis patients. Chemotherapy 1990; 36: 222-229. 5. 	 Verbist L. Dissociation of resistance between aztreonam. ceftazidime. cefepime. cefotaxime. and piperacillin in Gram negative isolates (multicentre study). 6th International Congress for Infectious Disease. Prague. Czech Republic 1994. 6. 	 Abb 1 .. Use of E-Test for susceptibility testing of Ps. aeruginosa." Mediterranean Congress of Chemotherapy. Athens. 1992. 7. 	 Sofianou DC. Vrenas S. Doumboyas 1. In-vitro susceptibility of clinical isolates of Pseudomonas aeruginosa to -lactam and aminoglycoside antibiotics . 1. Chemother 1989; 1 (6): 391-393. 8. 	 Nai-Xun Chin. Clynes N. Neu He. Resistance to Ciprofloxacin appearing during therapy. Am 1 Med 1989; 87 (Suppl5A): 28S-31S. 9. 	 Fung-Tomc 1. Kolek B. Bonner DP. Ciprofloxacin induced. low-level resistance to structurally unrelated antibiotics in Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus. Antimicrob Agents chemother 1993: 6: 1289-l296. The copyright of this article belongs to the Editorial Board of the Malta Medical Journal. The Malta Medical Journal’s rights in respect of this work are as defined by the Copyright Act (Chapter 415) of the Laws of Malta or as modified by any successive legislation. Users may access this full-text article and can make use of the information contained in accordance with the Copyright Act provided that the author must be properly acknowledged.  Further distribution or reproduction in any format is prohibited without the prior permission of the copyright holder. This article has been reproduced with the authorization of the editor of the Malta Medical Journal (Ref. No 000001)  

Abb 1 .. Use of E-Test for susceptibility testing of Ps. aeruginosa.&quot; Mediterranean Congress of Chemotherapy.

Dissociation of resistance between aztreonam. ceftazidime. cefepime. cefotaxime. and piperacillin in Gram negative isolates (multicentre study).

Doumboyas 1. In-vitro susceptibility of clinical isolates of Pseudomonas aeruginosa to -lactam and aminoglycoside antibiotics

Etest® technical guide no.3.

Major trends in the microbial aetiology of nosocomial infection.

Resistance to Ciprofloxacin appearing during therapy.

Wiora 1. Comparison of inhibitory and bactericidal activity of antipseudomonal antibiotics against Ps. aeruginosa isolates from cystic fibrosis patients. Chemotherapy

Antibiotic susceptibility patterns of local strains of Pseudomonas aeruginos

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Antibiotic susceptibility patterns of local strains of Pseudomonas aeruginos

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