Antibiotic susceptibility patterns among respiratory isolates of Gram-negative bacilli in a Turkish university hospital

Background Gram-negative bacteria cause most nosocomial respiratory infections. At the University of Cumhuriyet, we examined 328 respiratory isolates of Enterobacteriaceae and Acinetobacter baumanii organisms in Sivas, Turkey over 3 years. We used disk diffusion or standardized microdilution to test the isolates against 18 antibiotics. Results We cultured organisms from sputum (54%), tracheal aspirate (25%), and bronchial lavage fluid (21%). The most common organisms were Klebsiella spp (35%), A. baumanii (27%), and Escherichia coli (15%). Imipenem was the most active agent, inhibiting 90% of Enterobacteriaceae and A. baumanii organisms. We considered approximately 12% of Klebsiella pneumoniae and 21% of E. coli isolates to be possible producers of extended-spectrum beta-lactamase. K. pneumoniae isolates of the extended-spectrum beta-lactamase phenotype were more resistant to imipenem, ciprofloxacin, and tetracycline in our study than they are in other regions of the world. Conclusions Our results suggest that imipenem resistance in our region is growing.


Background
Nosocomial bacterial pneumonia is frequently polymicrobial, with gram-negative bacilli predominating [1]. Because delays in antimicrobial treatment can lead to adverse outcomes, the choice of empirical therapy is vital. Many effective antimicrobial agents are available, but the treatment of nosocomial pneumonia remains challenging. We recently reported the antibiotic-resistance patterns of respiratory isolates of Pseudomonas aeruginosa in our region [2]. The current study investigates the distribution and drug resistance of other gram-negative bacteria in the respiratory secretions of hospitalized patients. Table 1 and Table 2 present the antibiotic susceptibility patterns of our isolates. The most common organisms were Klebsiella spp (35%), A. baumanii (27%), and E. coli (15%). We also isolated rare organisms such as Stenotrophomonas maltophilia, Burkholderia spp, and Hafnia alvei. All studied Enterobacteriaceae (except Enterobacter spp) were far more susceptible to ticarcillin-clavulanate than to ticarcillin alone, which suggests that the primary mechanism of resistance in these organisms is β-lactamase production.

Results
K. pneumoniae accounted for 79% of Klebsiella isolates. Klebsiella spp were generally more susceptible to the tested antimicrobials than were Enterobacter spp, Serratia spp, or E. coli. The overall resistance rates to the third-generation cephalosporins (cefotaxime, ceftazidime, and ceftriaxone) were as follows: Klebsiella spp, 10%-19%; Serratia spp, 16%-33%; and Enterobacter spp, 22%-45%. Serratia spp were less resistant to third-generation cephalosporins than Enterobacter spp. E. coli isolates resistant to piperacillin, gentamicin, and the fluoroquinolones accounted for only 4% of all E. coli isolates. Imipenem was the most active agent against our isolates.
After imipenem, ciprofloxacin, and the aminoglycosides, tetracycline was the most active agent against A. baumanii. Tobramycin was more effective against A. baumanii than against Enterobacteriaceae. Tobramycin and imipenem were the most active agents against both gentamicin-and ciprofloxacin-resistant A. baumanii (Table 3).  We observed the ESBL phenotype in 10 E. coli isolates (20.8%) and 11 K. pneumoniae isolates (12.2%). All K. pneumoniae and E. coli isolates with the ESBL phenotype were resistant to tetracycline. Regarding K. pneumoniae isolates, 2 were susceptible to tobramycin, 3 to gentamicin, and 4 to ciprofloxacin, but 8 were susceptible to amikacin and imipenem. Regarding E. coli isolates, 4 were susceptible to tobramycin, 7 to gentamicin, 5 to ciprofloxacin, and 8 to amikacin, but all were susceptible to imipenem.  [7], and 51.9% in India [8].

Discussion
Although the isolation of Acinetobacter spp in respiratory specimens may reflect colonization and not necessarily infection [9], the most common site of nosocomial Acinetobacter infection is the lower respiratory tract, especially in mechanically ventilated patients [10]. Acinetobacter spp were the second most frequent gram-negative bacilli isolated from patients with pneumonia in Latin America [7]. In our survey, all compounds tested showed decreased activity among the A. baumanii isolates. Susceptibility to imipenem was >95% in Canada [11], India [8], and China [5]; susceptibility was 80.5% in our study and 55.5% in another study from Turkey [12]. The high prevalence of respiratory tract infections due to multiresistant A. baumanii will stimulate the use of carbapenems and possibly increase carbapenem resistance in our region.
In their study, no Enterobacter or Serratia isolates were resistant to imipenem. In our study, however, the rates of susceptibility to imipenem were 86.2% for Enterobacter spp and 76.5% for Serratia spp. Imipenem susceptibility for these two species was >95% in other parts of the world [7][8][9][10]13]. Moreover, Serratia spp were at least 95% susceptible to ceftazidime in the United States [14], Canada [11], India [8], China [5], and Korea [6]. We found only one imipenem-resistant E. coli isolate. It was resistant to ampicillin, ticarcillin, and piperacillin but susceptible to ceftazidime, ceftriaxone, and aztreonam. This profile suggests an oxacillinase with carbapenemase properties. This finding is interesting because class D enzymes have been found only in Acinetobacter spp [16]. Two imipenem-resistant Klebsiella spp were resistant to all β-lactams, including aztreonam. These species were probably expressed a metallo-β-lactamase with additional mechanisms (efflux, cephalosporinase hyperproduction) [16].
The absence of a confirmation test for the ESBL phenotype limits the impact of our results. On the other hand, it is known that supplemented media (blood) can alter the zone diameters for several agents and bacterial species. Despite these limitations, our data can be used for local therapeutic choices.

Conclusions
We previously presented the antibiotic susceptibility patterns of 249 respiratory isolates of P. aeruginosa during the same period [2]. When combined with our current data, these results show that, in our region, ceftazidime can still be used for managing respiratory infections due to gramnegative aerobic bacteria in combination with aminoglycosides. It appears that increasing imipenem resistance may cause serious therapeutic problems in future.

Methods
We collected our data from 01/01/1999 to 01/01/2002 at the microbiology laboratory of the University of Cumhuriyet. We processed the data to eliminate duplicate registrations. We excluded any isolates collected within 7 days when they came from the same specimen source of the same patient. We initially identified the isolates using such routine methods as colonial/microscopic morphology and enzymatic characteristics. We confirmed species identification with API-bioMerieux products. We retrospectively analyzed antibiotic susceptibility patterns in 238 respiratory isolates of Enterobacteriaceae members and 90 respiratory isolates of A. baumanii. We accepted all consecutive isolates because we did not attempt to distinguish actual pathogens from colonizing strains. Specimen types consisted of sputum (54.2%), transtracheal/ endotracheal aspirates (24.6%), and bronchial lavage fluid (21.2%). We cultured sputum samples that showed no oral contamination in the presence of sputum purulence or a suspected lower respiratory infection.
We confirmed susceptibility to 18  disk zone sizes; thereafter, we used a coordinating laboratory to determine the minimal inhibitory concentrations (MICs) of these 18 antimicrobial agents, accomplished with a standardized microdilution technique (Sceptor System, Becton Dickinson Microbiology System). We used this system to determine MICs for all strains. We used the NCCLS criteria to identify possible extendedspectrum β-lactamase (ESBL)-producing strains of Klebsiella spp and E. coli when MICs were increased (2 mg/mL) with ceftazidime and/or ceftriaxone and/or aztreonam [18], but we lacked a test to confirm the ESBL phenotype.
We classified our results into two categories. We labeled strains deemed susceptible by the disk diffusion method or microdilution technique as susceptible. We labeled all resistant and intermediate isolates as resistant. We divided the number of resistant isolates by the total number of isolates that had undergone susceptibility testing.

Authors' contributions
UG had primary responsibility for study design, collection of data, and writing the manuscript. IA, MZB, TE had intellectual contribution as well as the writing of manuscript. All authors read and approved the final manuscript.