International Journal of Antimicrobial Agents 17 (2001) 465– 469 www.ischemo.org Original article Antimicrobial resistance and serotype distribution of Streptococcus pneumoniae isolates from Crete, Greece Sofia Maraki *, Athanasia Christidou, Yannis Tselentis Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Uni6ersity Hospital of Crete, 711 10 Heraklion, Crete, Greece Received 28 November 2000; accepted January 2001 Abstract Susceptibility to 14 antibiotics was determined for 125 clinical isolates of Streptococcus pneumoniae collected over a 3-year period in Crete, Greece Twenty-three isolates (18.4%) showed intermediate resistance and 15 (12%) high-level resistance to penicillin Erythromycin, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazole and sparfloxacin resistance rates were 16.8, 10.4, 19.2, 24.8 and 9.6%, respectively Multiple resistance was observed in 22 strains Vancomycin and levofloxacin were the most active agents tested The most prevalent serotype among penicillin-susceptible pneumococci was 14, followed by 9, and 1, while among penicillin-intermediate or -resistant strains serotype 23 was predominant followed by 19 and These results show that as well as a high level of penicillin resistance in this region, some strains are also resistant to other antibiotics and may show multi-drug resistance © 2001 Elsevier Science B.V and International Society of Chemotherapy All rights reserved Keywords: Antibiotic susceptibility; Serotypes; Streptococcus pneumoniae Introduction Materials and methods Streptococcus pneumoniae is the leading cause of bacterial pneumonia, acute otitis media, and sinusitis and the most frequent aetiological agent of bacterial meningitis and bacteraemia [1,2] Resistance of S pneumoniae to b-lactam agents and antimicrobial agents is increasing worldwide [3,4] The continued frequency and severity of pneumococcal infections and the increasing rates of resistance to antimicrobial agents means there is need for continuous local surveillance as the prevalence and serotype of resistant strains may vary from one geographical area to another The present study, which is the first in our area, describes the antimicrobial susceptibilities and serotypes of S pneumoniae strains isolated from clinical specimens in Crete, Greece, from 1997 to 1999 2.1 Bacterial isolates * Corresponding author Present address: 1, Delphon street, 713 05 Heraklion, Crete, Greece Tel.: +30-81-392598; fax: + 30-81-392597 E-mail address: sv9sk@danae.med.uoc.gr (S Maraki) Consecutive S pneumoniae isolates recovered from clinical specimens submitted to the Laboratory of Clinical Microbiology, at the University Hospital of Crete, were collected over a 3-year period (1997 – 1999) Only a single isolate from each patient was included in the study Pneumococci were identified using standard techniques, including Gram stain characteristics, colonial morphology, optochin susceptibility and bile solubility [5] 2.2 Susceptibility testing Susceptibility testing used the agar dilution method described by the National Committee for Clinical Laboratory Standards [6,7] The following antibiotics were tested: penicillin, cefuroxime, ceftriaxone, cefepime, 0924-8579/01/$20 © 2001 Elsevier Science B.V and International Society of Chemotherapy All rights reserved PII: S - ( ) 0 3 - 466 S Maraki et al / International Journal of Antimicrobial Agents 17 (2001) 465–469 Fig Frequency of occurrence of PSP, PIR and PRP strains isolated between 1997 and 1999 imipenem, meropenem, erythromycin, clarithromycin, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazole, levofloxacin, sparfloxacin and vancomycin The MIC was taken as the lowest concentration of antibiotic that inhibited growth In all our studies, S pneumoniae ATCC 6305 (penicillin-susceptible) and S pneumoniae ATCC 49619 (penicillin-resistant) were used as control strains 2.3 Serotyping Serotyping was performed by the capsular swelling method with specific antisera available from the Statens Seruminstitute of Copenhagen, Denmark Results One hundred and twenty five strains were collected over the year period The origin of these was blood (n =18), cerebrospinal fluid (n =4), middle ear aspirates (n =10), ocular secretions (n= 6), upper respiratory tract specimens (n =39), lower respiratory tract specimens (n =45) and other sites (n= 3) Of these strains, 87 (69.6%) were susceptible to penicillin (PSP), 23 (18.4%) were intermediate (PIR) and 15 (12%) had high level resistance to penicillin (PRP) The percentage of isolates with intermediate and high level resistance to penicillin increased over the study period (Fig 1) Combined intermediate and high level resistance rates were 26.2% in 1997, 31.1% in 1998 and 34.2% in 1999 However, the proportion of highly resistant isolates in 1999 was considerably increased over the proportion found in the preceding years of the study (Fig 1) The relationship between penicillin activity and the activity of other b-lactam agents and additional antimicrobial agents is shown in Table Resistance to these agents was more common among penicillin-intermediate (PIR) strains, than among PSP strains and was most common among highly PRP organisms It is important to note that all PSP pneumococcal isolates were also susceptible to other b-lactam agents tested Isolates were uniformly susceptible to levofloxacin and vancomycin There was a higher incidence of PRP strains in specimens from children (44.5%) compared with those recovered from adults (19.7%) Isolates from the middle ear aspirates were associated with higher resistance rates to penicillin than those recovered from other body sites (Table 2) Resistance rates observed to other antibiotics were: erythromycin (16.8%), chloramphenicol (10.4%), tetracycline (19.2%), erythromycin (16.8%), trimethoprimsulphamethoxazole (24.8%) and sparfloxacin (9.6%) Among PSP strains serotype 14 was predominant (20.63%), followed by (19.05%), (17.47%), Table Analysis of resistance of 125 strains of S pneumoniae to 13 antimicrobial agents, according to their susceptibilities to penicillin % of PSP isolates (n = 87) % of PIR isolates (n = 23) % of PRP isolates (n =15) Antimicrobial agent S I R S I R S I R Cefuroxime Ceftriaxone Cefepime Imipenem Meropenem Erythromycin Clarithromycin Chloramphenicol Tetracycline Trimethoprim-sulphamethoxazole Levofloxacin Sparfloxacin Vancomycin 98.9 100 100 100 100 87.4 87.4 98.9 94.3 95.4 100 89.7 100 1.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 10.3 0.0 0.0 0.0 0.0 0.0 0.0 12.6 12.6 1.1 5.7 4.6 0.0 0.0 0.0 47.8 87 78.3 87 100 82.6 82.6 65.2 52.2 39.1 100 95.7 100 4.4 13 21.7 13 0.0 0.0 0.0 0.0 0.0 17.4 0.0 4.3 0.0 47.8 0.0 0.0 0.0 0.0 17.4 17.4 34.8 47.8 43.5 0.0 0.0 0.0 6.7 26.7 13.3 13.3 60 60 60 60 46.7 20 100 100 100 0.0 53.3 60 73.4 40 0.0 0.0 0.0 0.0 20 0.0 0.0 0.0 93.3 20 26.7 13.3 0.0 40 40 40 53.3 60 0.0 0.0 0.0 S Maraki et al / International Journal of Antimicrobial Agents 17 (2001) 465–469 Table Sources of 125 S pneumoniae isolates recovered from clinical specimens between 1997 and 1999 Specimen origin No (%) of isolates that were: Upper respiratory tract Lower respiratory tract Blood CSF Ocular secretions Middle ear aspirates Miscellaneous Total (%) PRP Total no of Isolates PSP PIR 22 11 39 35 45 17 1 0 18 10 87 (69.6%) 23 (18.4%) 15 (12%) 125 (6.35%), 4, 8, 15, 18 (4.76% each), 6, 10, 17 (3.17% each) and 3, 11, 12, 19, 23 (1.59% each) There was no difference in distribution of pneumococcal serotypes in PSP strains over the study period However, serotype 23 was the most prevalent serotype among PIR and PRP strains followed by 19, and Table shows the yearly distribution of serotypes among PIR and PRP strains Resistance to multiple (at least three) antimicrobial agents was also observed among 22 (57.9%) PIR and PRP strains Multi-resistant strains were more frequently associated with serotypes 23 (13 strains) and 19 (six strains); there was just one multi-resistant strain of serotypes 6, 9, and 14 Table Serotypes of 38 PIR and PRP clinical strains isolated between 1997 and 1999 No (%) of strains Serotype 23 19 14 Total 13 12 38 (34.2) (31.6) (15.8) (10.5) (5.3) (2.6) (100) No of strains belonging to each serotype isolated in: 1997 1998 1999 – – – 11 – 14 2 – 13 467 Discussion Pneumococcal strains with decreased susceptibilities to penicillin emerged in the late 1960s and 1970s in Australia, South Africa and the US and then spread worldwide [8 –11] In Europe, countries with high rates of isolation of penicillin-resistant strains include Spain, France, Hungary, Romania and Bulgaria [12– 15] Higher rates of resistance have been reported from Hungary (58%) [16] This is assumed to be due to the frequent administration of antibiotics that leads to nasopharyngeal colonization and infection with resistant pneumococcal stains Switzerland, the Netherlands, Great Britain, and Sweden report low rates of pneumococcal resistance [17–20] In Greece, two recent studies, one conducted in Athens and the other in southwestern Greece, reported results similar to our own suggesting that there are no regional differences within Greece [21,22] However, lower resistance rates to penicillin (14%) have been previously reported by other investigators [23] This is the first study undertaken in Crete, an island at the most southern point of Greece Our results show a gradual increase in the rate of isolation of PRP pneumococcal stains during the years of the study These findings show the importance of continuous monitoring of S pneumoniae strains for antimicrobial resistance in this area The mechanism of resistance to penicillin is related to the presence of altered penicillin-binding proteins (PBPs) that have decreased affinity for penicillin and other b-lactam antibiotics [24] Therefore, strains of intermediate or high resistance to penicillin would also exhibit reduced susceptibility to other b-lactam agents In fact, in the present study we have found that all penicillin susceptible pneumococcal isolates were also susceptible to the cephalosporins tested, while a high level of resistance to these agents occurred in penicillin-resistant isolates Resistance among pneumococci was not restricted to only b-lactam agents Erythromycin-resistant isolates (16.8%) were uniformly cross-resistant to clarithromycin Resistance to macrolides, which are used as alternative drugs for the treatment of pneumococcal infections, is mainly constitutive, MLSB type [25] Our resistance rates to macrolides are comparable to those of other investigators [26,27] PRP strains showed higher rates of resistance to chloramphenicol, tetracycline and trimethoprim-sulphamethoxazole and these results are similar to those previously reported in other countries [28,29] Twenty-two of the 38 penicillin-resistant strains were also resistant to multiple antibiotics This is similar to reports from other countries with increasing rates of isolation of multidrug-resistant pneumococci and who showed that PRP isolates also carry genetic determinants encoding resistance to multiple antibiotics [17,30] 468 S Maraki et al / International Journal of Antimicrobial Agents 17 (2001) 465–469 All the 125 S pneumoniae isolates were susceptible to levofloxacin Previous studies have shown the good in-vitro activity of the drug against S pneumoniae [31,32] Additionally, clinical investigations have shown its effectiveness in the treatment of pneumococcal respiratory tract infections [33,34] Vancomycin had excellent in-vitro activity against all isolates tested However, this antibiotic must be retained for the treatment of severe infections caused by S pneumoniae strains resistant to other antimicrobial agents The incidence of PRP S pneumoniae strains in severe invasive infections such as septicaemia and meningitis, was very low compared with those from aural infections and infections of the respiratory tract Such resistant microorganisms causing otitis media, sinusitis or pneumonia may spread and cause invasive infections that are difficult to treat Infections with PIR and PRP strains occurred more frequently in children under years of age This is attributed to the frequent administration of antibiotics either for treatment of infections or as prophylaxis The 38 resistant pneumococcal isolates serotyped, were found to belong to five serogroups: 23, 19, 9, 14, 6, and 15 (in order of decreasing frequency) These serogroups are included in the 23-valent polysaccharide pneumococcal vaccine, indicating the usefulness of the vaccine to the Cretan population S pneumoniae strains belonging to serogroup 23 were common during 1997, while those belonging to serogroup 19 were more frequently isolated during 1998 and 1999 All 13 strains belonging to serogroup 23 were also resistant to chloramphenicol, tetracycline and trimethoprim-sulphamethoxazole This resistance profile has already been seen in strains belonging to serogroup 23 isolated in Spain and the US [12,35,36] In conclusion, our data indicate the presence of penicillin resistance and multiple resistance among clinical isolates of S pneumoniae from Crete Further studies to define the genetic relatedness of the isolates by pulsed field gel electrophoresis are in progress As resistance patterns are changing and as they vary among geographical locations, continuous surveillance of changes in the susceptibilities of antibiotics used to treat pneumococcal infections is necessary for determining therapeutic regimens References [1] Musher DM Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity and treatment Clin Infect Dis 1992;14:801 – [2] Musher DM Streptococcus pneumoniae In: Mandell GL, Bennett JE, Dolin R, editors Principles and practice of infectious diseases New York: Churchill Livingstone, 1995:1811 – 26 [3] Baquero F Pneumococcal resistance to b-lactam antibiotics: a global geographic overview Microb Drug Resist 1995;1:115 – 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Antibiotic resistance of clinical isolates of Streptococcus pneumoniae in Greece J Clin Microbiol 1994;32:3056 – [24] Grebe T, Hakenbeck R Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae. .. with serotypes 23 (13 strains) and 19 (six strains); there was just one multi-resistant strain of serotypes 6, 9, and 14 Table Serotypes of 38 PIR and PRP clinical strains isolated between 1997 and. .. isolated in Spain and the US [12,35,36] In conclusion, our data indicate the presence of penicillin resistance and multiple resistance among clinical isolates of S pneumoniae from Crete Further