Comp Clin Pathol DOI 10.1007/s00580-014-1937-3
ORIGINAL ARTICLE
Resistance to different generations of quinolones in Streptococcus pneumoniae strains isolated from hospitals in Shiraz Mohammad Kargar & Fataneh Moein Jahromi & Abbas Doosti & Zahra Mohammadalipour & Shahrokh Lorzadeh
Received: 25 October 2013 / Accepted: 29 April 2014 # Springer-Verlag London 2014
Abstract Streptococcus pneumoniae is a leading cause of respiratory tract infections, including pneumonia. In recent years, due to the incidence of resistance to commonly used antibiotics, quinolones have been taken into consideration in treatment of S. pneumoniae. The present study aimed to investigate the quinolone resistance rate among pneumococcus isolates from Shiraz. This cross-sectional-descriptive study was performed on 45 strains of S. pneumoniae isolated from clinical samples. Initially, using biochemical tests and PCR technique with specific primers of lytA gene, S. pneumoniae strains were identified. Subsequently, antibiotic resistance to quinolones was evaluated by CLSI criteria. Among 45 strains isolated from clinical specimen, resistance to nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin and levofloxacin were estimated 88.22, 73.43, 53.33, 48.88 and 42.22 %, respectively. The highest frequency of resistance corresponded to the people with pneumonia. Besides, the greatest resistance to all antibiotics related to the age group of 31–40. Statistical analysis indicated no significant association between antibiotic resistance and disease type or age groups. In concurrence with other investigations, our results showed that the level of resistance to quinolones is increasing in Shiraz. Hence, the quinolone resistance in this region is a serious issue; therefore, the regional pattern of antimicrobial resistance on therapeutic regimes must be taken into consideration.
M. Kargar (*) : F. M. Jahromi : Z. Mohammadalipour Department of Microbiology, Islamic Azad University, Jahrom Branch, Jahrom, Iran e-mail:
[email protected] A. Doosti : S. Lorzadeh Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Keywords Streptococcus pneumoniae . Quinolones . Antibiotic resistance . lytA
Introduction Streptococcus pneumoniae is an important human pathogen which is responsible for respiratory diseases such as pneumonia and other serious diseases including meningitis and otitis media. The international concern about penicillin resistance or multidrug-resistant (MDR) strains of S. pneumoniae led to the development and consumption of quinolones, an advanced antipneumococcal drug (Pan et al. 2001). These drugs are analogues of nalidixic acid which was favoured in the 1960s (Lesher et al. 1962). Fluoroquinolones, another generation of quinolones including ciprofloxacin and ofloxacin, are extremely important in health care in view of their broad antimicrobial spectrum and effectiveness in a variety of infectious diseases through the oral route. In addition, they relatively cause less side effects, and resistance to these antibiotics do not spread quickly. Generally, quinolones contain a carboxylic acid at position 3 of naphthyridine ring, and fluoroquinolones comprise an additional fluorine atom at position 6 of the main ring and also many of them have a piperazine ring at position 7 (Foroumadi et al. 1998). Previous research indicates that the penetration of the quinolones into cells extensively depends on the nature of the substituent at C7. Besides, the only position at which the molecule can handle a large substituent is position 7 (Mandell 1990). Many vigorous quinolones have been reported with changes in position 7; thus, C7 of the quinolone core has adrastic influence on antibiotic characteristics (Siporin 1989). Mostly, quinolones prescribed in unique situations where antibiotic resistance has been developed (Sanders et al. 1984). Quinolones target two essential bacterial enzymes, DNA gyrase and topoisomerase IV (Balsalobre et al. 2011; Patel et al. 2010). They have
Comp Clin Pathol
been extensively utilized for pneumococcal infections, but later few resistant strains arose in different parts of the world. Following the first resistance report in 1998 from Canada, throughout the last three decades the prevalence of resistance to quinolones has been increasing rapidly (Adam et al. 2007). A mutation in the bacterial chromosome is responsible for resistance to quinolones, though; any plasmid-mediated quinolone resistance has not been reported (Smith 1984). The occurrence of S. pneumoniae varies in different areas, and then requires continuous monitoring. Thus, the aim of this study was to investigate the prevalence of quinolone-resistant strains of S. pneumoniae, isolated from two main hospitals in Shiraz.
performed in 25-μl volume containing, 1 μl of template DNA, each primer1 μl, MgCl21 μl, dNTPs 0.5 μl and Taq polymerase enzyme 0.25 μl. PCR procedure is demonstrated in Table 1. Amplified products were visualized with ethidium bromide on 1 % agarose gel by UV transilluminator. Estimation of susceptibility to quinolones
Meterials and methods
In order to assess the bacterial susceptibility to three generations of quinolone family, antibiogram test was carried out by standard Clinical and Laboratory Standards Institute (CLSI) procedures. Antibiotic susceptibility rate of the colonies to nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), ofloxacin (5 μg) and levofloxacin (5 μg) was estimated by measuring the diameter of the zone of inhibition, according to manufacturer’s directions (Denmark, ROSCO).
Bacterial strain
Statistical analysis
This cross-sectional-descriptive study was performed on 45 strains of S. pneumoniae isolated from the patients admitted to intensive care units (ICU) of Nemazee and Shahid Faghihi Hospitals in Shiraz from 2011 to 2012. Clinical samples were composed of sputum, blood and cerebrospinal fluid, collected from patients with pneumonia, meningitis and unknown origin fever, while all patients aged between 10 to 60 years old.
Data were analyzed by SPSS version 17 (SPSS Inc., Chicago, IL, USA) and Fisher’s exact test. P value less than 0.05 was considered statistically significant.
Biochemical identification Samples were cultured on blood agar medium containing 5 % sheep blood agar and incubated for 24 h at 37 °C, in order to isolate S. pneumoniae strains. Basic identification of the colonies carried out using colony characteristics, type of hemolysis, Gram staining, bile solubility and Optochin test. PCR of lytA gene
Results From 45 studied clinical isolates, 29 (64.45 %) samples belonged to men, and 16 (35.55 %) to women. Furthermore, 23 samples (51.11 %) pertained to the patients with pneumonia, 16 (35.55 %) to patients with meningitis, and 7 (15.55 %) to those who had fever with unknown origin. All patients aged between 10 and 58 years old, while most of the them were in the age group of 31–40 years with frequency of 19 (42.22 %) and the least with a frequency of 4 (8.88 %) between 51– 60 years. S. pneumoniae strains were identified by PCR technique with specific primers of F-lytA and R-lytA, giving 295 bp fragments (Fig. 1). Applying the standard disk
In this study, bacterial DNA was extracted by boiling method and stored in −20 °C as stock. In order to verify the accuracy of identified strains, using PCR technique, lytA gene was amplified using specific forward and reverse primers (Fukushima et al. 2008) (Table 1). Primers were obtained from the CinnaGene, Co., Tehran, Iran. PCR amplification was Table 1 PCR conditions for lytA gene amplification Primers Primer sequence (5′→3′) lytA-F lytA-R
Amplicon PCR conditions size, bp
CAACCGTACAGA 295 bp ATGAAGCGG TTATTCGTGCAA TACTCGTGCG
8 5min at 94C < 1min at 94C 32 cycles 1min at 58C : 1min at 72C 8 min at 72C
Fig. 1 Products of lytA gene amplification with 295 bp fragments; M 100 bp DNA marker, 1 positive control, 2 negative control, 3–6 confirmed samples
Comp Clin Pathol Fig. 2 Illustration of susceptibility rate of clinical samples to quinolone antibiotics
Levofloxacin Norfloxacin Ofloxacin Ciprofloxacin Nalidixic acid 0%
diffusion methods, estimated resistance rate to antibiotics; nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin and levofloxacin were 82.22, 73.43, 53.33, 48.88 and 42.22 %, respectively (Fig. 2). Moreover, the highest rate of resistance to all antibiotics corresponded to the age group of 31–40 years (Table 2), while no significant correlation was found between the age group and antibiotic resistance. Furthermore, our results indicated that there was not any significant correlation between resistance to utilized quinolones and the type of diseases (Table 3).
Discussion S. pneumoniae is one of the major roots of acute respiratory infections resulting in 500 thousand cases of pneumonia, 7 million cases of otitis media and 3 to 7 million deaths each
Table 2 Antibiotic resistance results for clinical specimens according to age groups (n=45)
20% 40% susceptible
60% 80% 100% semi-resistant resistant
year (Kargar et al. 2012). In the late 1990s, quinolone-resistant strains spread throughout the world. Geographically rates of quinolone-resistant strains have been variable and rising in recent years. The level of resistance to quinolones in all European countries is low except Poland (4.4 %), Finland (6.6 %), Spain (7 %) and Italy (7.2 %), while the highest rate belongs to Canada. So that the resistance level of 0.6 % in 1998 raised up to 7.3 % in 2006 which is constantly increasing significantly (Linares et al. 2010). Quinolone resistance level in Asian countries is higher than in other parts of the world (Fuller and Low 2005), for instance, resistance level in Korea, Philippines, Sri Lanka and Hong Kong are 6.1, 9.1, 9.5 and 14.3 %, respectively (Yamamoto et al. 2009). In addition, the prevalence of levofloxacin-resistant strains will be a major concern in Middle East countries including Lebanon, Kuwait and Qatar (Naba et al. 2010). Experiments performed in Canada during 1997–2005 and southern Nigeria in 2010,
Antibiotic
10–20
21–30
31–40
41–50
51–60
Total
P value
Nalidixic acid Ciprofloxacin Norfloxacin
5 (11.11) 5 (11.11) 3 (6.67)
10 (22.22) 10 (22.22) 9 (20)
18 (40) 18 (40) 18 (40)
5 (11.11) 4 (8.89) 4 (8.89)
3 (6.67) 3 (6.67) 3 (6.67)
41 (91.11) 40 (88.89) 37 (82.22)
0.151 0.080 0.468
Levofloxacin Ofloxacin
3 (6.67) 4 (8.89)
6 (13.33) 10 (22.22)
18 (40) 18 (40)
3 (6.67) 4 (8.89)
1 (2.22) 3 (6.67)
31 (68.89) 39 (86.66)
0.318 0.276
Table 3 Antibiotic resistance results for clinical specimens according to disease type (n=45) Antibiotic
NA
CP
NOR
OFL
LEV
Pneumonia Meningitis Fever
22 (48.89) 14 (31.11) 5 (11.11)
21 (46.67) 14 (31.11) 5 (11.11)
20 (44.44) 13 (28.89) 4 (8.89)
21 (46.67) 13 (28.89) 5 (11.11)
18 (40) 10 (22.22) 3 (6.67)
NA nalidixic acid, CP ciprofloxacin, NOR norfloxacin, OFL ofloxacin, LEV levofloxacin
Comp Clin Pathol
suggested that the highest level of quinolone resistance corresponded to ciprofloxacin and the lowest resistance was to levofloxacin, while most of the resistant strain was isolated from adult patients (Adam et al. 2007; El-Mahmood et al. 2010; Rodriguez et al. 2011; Orr et al. 2010). Our results as well as other studies indicated that resistance to quinolones is increasing substantially. Nalidixic acid and ciprofloxacin reported to have the highest resistance rate while levofloxacin the least. The divergent resistance rate at different parts of the world is believed to be associated with the differences in geographic location, clinical features and differences in the advancement of antibiotic consumption rates in each country. Moreover, similar to other researches, most people with resistant infections to quinolones were adults, and it has been minimal in children. This can be explained in two ways; first, quinolones such as levofloxacin and ciprofloxacin are not prescribed for children; secondly, adults are at greater risk to confront resistant strains due to more interactions with the community.
Conclusion According to the results, quinolone resistance among S. pneumoniae strains is expanding in the area of research. The results indicate that the highest resistance corresponded to nalidixic acid (first generation quinolones) and the least to levofloxacin (third generation quinolones). Therefore, the prevention of excessive use of quinolone antibiotic family and further research on molecular mechanisms of resistance to these antibiotics are recommended. Acknowledgments The authors are grateful to the Islamic Azad University of Jahrom and Shahrekord, for their executive support of this project.
References Adam HJ, Schurek KN, Nichol KA, Hoban CJ, Baudry TJ, Laing NM (2007) Molecular characterization of increasing fluoroquinolone resistance in Streptococcus pneumoniae isolates in in Canada, 1997 to 2005. Antimicrob Agents Chemother 51(1):198–207 Balsalobre L, Ferrandiz MJ, Alba G, De la Campa AG (2011) Nonoptimal DNA topoisomerases allow maintence of supercoiling level and improve fitness of Streptococcus pneumoniae. Antimicrob Agents Chemother 55(3):1079–1105 El-Mahmood A, Isa H, Mohammed, Tirmidhi AB (2010) Antimicrobial susceptibility of some respiratory tract pathogens to commonly used antibiotics at the Specialist Hospital, Yola, Adamawa State, Nigeria. J Clin Med Res 2(8):135–142
Foroumadi AR, Haghighat P, Emami S, Moshafi MH (1998) Synthesis of new piperazinyl quinolones and investigation of their in vitro antibacterial activities. J Kerman Univ Med Sci 5(3):103–109 Fukushima KY, Yanagihara K, Hirakata Y, Sugahara K, Morinaga Y, Kohno S (2008) Rapid identification of penicillin resistance genes and simultaneous quantification of Streptococcus pneumoniae in purulent sputum samples by us of a novel real-time multiplex PCR assay. J Clin Microbiol 46(7):2384–2388 Fuller JD, Low DE (2005) A review of Streptococcus pneumoniae infection treatment failures associated with fluoroquinolone resistance. Clin Infect Dis 41(1):118–121 Kargar M, Baghernejad M, Ghorbani-daline S, Hashemizade Z (2012) Evaluation of molecular mechanisms resistance to macrolides by Streptococcus pneumoniae strains isolated from Nemazee and Shahid Faghihi Hospitals in Shiraz. Sci J Kurdistan Univ Med Sci 16(4):83–91 Lesher GY, Fredrich EJ, Greott MD, Bailey JH, Brundach PR (1962) 1,8Naphthyridine derivatives, a new class of chemotherapeutic agents. J Med Chem 91:1063–1068 Linares J, Ardauny C, Pallares R, Fenoll A (2010) Changes in antimicrobial resistance serotypes and genotypes in Streptococcus pneumoniae over a 30-year period. Clin Microbiol Infect 16(5): 402–410 Mandell GL (1990) In: Hardman JG, Limbird LF, Molinoff PB, Ruddon RW (eds) Goodman and Gilmans the pharmacological basis of therapeutics, 8th edn. Pergamon Press Inc, New York Naba MR, Araj GF, Baban TA, Tabbarah ZA, Awar GN, Kanj SS (2010) Emergence of fluoroquinolone-resistant Streptococcus pneumoniae in Lebanon: a report of three cases. J Infect Public Health 3(3):113– 117 Orr D, Wilkinson P, Moyce L, Martin S, George R, Pichon R (2010) Incidence and epidemiology of levofloxacin resistance in Streptococcus pneumonia: experience from a tertiary referral hospital in England. J Antimicrob Chemother 65(3):449–452 Pan XS, Yague G, Fisher LM (2001) Quinolone resistance mutations in Streptococcus pneumoniae GyrA and ParC proteins: mechanistic insights into quinolone action from enzymatic analysis, intracellular levels, and phenotypes of wild-type and mutant proteins. Antimicrob Agents Chemother 45(11):3140–3147 Patel S, Melano R, Mcgeer A, Greea K, Low DE (2010) Characterization of the quinolone resistant determining regions in clinical isolates of Pneumococci collected in Canada. Ann Microbiol Antimicrob 9(3): 1–6 Rodriguez I, Ramos B, Rios E, Cercenado E, Ordobas M, Carlos Sanz J (2011) Clonal spread of levofloxacin-resistance Streptococcus pneumoniae invasive isolates in Madrid, Spain, 2007 to 2009. Antimicrob Agents Chemother 55(5):2469–2471 Sanders CC, Sanders WE, Georing RV, Werner V (1984) Selection of multiple antibiotic resistance by quinolones, Blactams and aminoglycosides with special reference to cross resistance between unrelated drug-classes. Antimicrob Agents Chemother 26(6):797–801 Siporin C (1989) The evolution of fluorinated quinolones: pharmacology, microbiological activity, clinical uses and toxicities. Annu Rev Microbiol 43:601–627 Smith JT (1984) Mutational resistance to 4 quinolone antibacterial agents. Eur J Clin Microbiol 3(4):347–350 Yamamoto K, Yanagihara K, Sugahara K, Imamura Y, Seiki M, Izumikawa K (2009) In vitro activity of garenoxacin against Streptococcus pneumonia mutants with characterized resistance mechanisms. Antimicrob Agents Chemother 53(8):3573–3575
