Dorota Tichaczek-Goska, Dorota Wojnicz
Synergistic Effect of Normal Human Serum
and Antimicrobial Agents Against Colistin-resistant
Citrobacter freundii
Strains*
Synergistyczne działanie normalnej surowicy ludzkiej oraz antybiotyków
wobec szczepów
Citrobacter freundii
opornych na kolistynę
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
Abstract
Background. The bactericidal action of normal human serum (NHS) is an important mechanism protecting the host organism against infections caused mainly by Gram-negative bacteria such as strains of the Citrobacter genus. Owing to the complex structure of their outer membrane, many bacterial strains are resistant to complement-dependent host defense. Although this phenomenon is rare within Citrobacter spp. strains, serum-resistant bacilli have been found. Antibiotic resistance among bacteria belonging to Enterobacteriaceae is presently a common and serious epidemiological problem.
Objectives. The scope of this study was to investigate the combined effect of NHS and antimicrobial agents against clinical colistin-resistant Citrobacter freundii strains.
Material and Methods. The study was carried out on C. freundii strains isolated from bronchoalveolar lavage. The bactericidal activity of NHS and its combinations with antibiotics at subinhibitory concentrations was deter-mined.
Results. The survival of colistin- and serum-resistant C. freundii strains incubated in the suspension of NHS and ½ MICs of colistin was significantly reduced. The combination of NHS with amikacin and colistin sublethal concen-trations demonstrated strong bactericidal effects against C. freundii 1553, but not against C. freundii 1338. Conclusions. The susceptibility to NHS combined with colistin and amikacin at subinhibitory concentrations can vary between bacteria of the same species (Adv Clin Exp Med 2011, 20, 2, 131–136).
Key words: synergistic effect, colistin, amikacin, human serum.
Streszczenie
Wprowadzenie. Bakteriobójcze działanie normalnej surowicy ludzkiej (NSL) jest ważnym czynnikiem chronią-cym organizm gospodarza głównie przed zakażeniami wywoływanymi przez bakterie Gram-ujemne, m.in. pałeczki z rodzaju Citrobacter. Wiele bakterii charakteryzuje się opornością na mechanizmy obronne organizmu żywiciela, co jest związane ze złożoną strukturą błony zewnętrznej drobnoustrojów. Oporność na antybiotyki wśród szcze-pów z rodziny Enterobacteriaceae stanowi poważny problem epidemiologiczny.
Cel pracy. Określenie synergistycznego działania NSL oraz wybranych antybiotyków wobec klinicznych szczepów
Citrobacter freundii.
Materiał i metody. Badania przeprowadzono na szczepach C. freundii wyizolowanych z popłuczyn oskrzelowo-pę-cherzykowych. Określono bakteriobójcze działanie NSL w połączeniu z podprogowymi stężeniami antybiotyków. Wyniki. Przeżywalność szczepów bakteryjnych opornych zarówno na NSL, jak i kolistynę została ograniczona pod wpływem NSL oraz ½ MIC kolistyny. Ponadto wykazano zwiększoną podatność szczepu C. freundii 1553 na łączne działanie NSL oraz podprogowych stężeń kolistyny i amikacyny. Zjawiska tego nie odnotowano natomiast w przypadku szczepu C. freundii 1338.
Wnioski. Podatność bakterii na skojarzone działanie NSL i antybiotyków w stężeniach podprogowych może różnić się między szczepami w obrębie tego samego gatunku (Adv Clin Exp Med 2011, 20, 2, 131–136.
Słowa kluczowe: synergizm, kolistyna, amikacyna, ludzka surowica.
Adv Clin Exp Med 2011, 20, 2, 131–136 ISSN 1230-025X
OrIGINAL PAPErS
© Copyright by Wroclaw Medical University
Citrobacter freundii strains are Gram-negative rods belonging to the family Enterobacteriaceae
and are found in water, feces and urine. They can occur in alimentary infections and often are the cause of opportunistic and nosocomial infections.
C. freundii bacilli have been also associated with pneumonia, bacteriemia, neonatal meningitis and brain abscess [1, 2].
The bactericidal activity of serum is an imptant defense mechanism protecting the host or-ganism against infections caused by a wide range of microorganisms, including bacteria belonging to the Enterobacteriaceae family. Many bacterial strains are resistant to complement-dependent host defense, and this feature is related to the complex structure of their outer membrane e.g. capsule, outer membrane proteins or lipopoly-saccharides [3–6]. The bacterial susceptibility to sera can be increased in the presence of sub-in-hibitory concentrations of certain antimicrobial agents [7–9].
Amikacin (AN) is an aminoglycoside anti-biotic which acts by binding to the bacterial 30S ribosomal subunit and inhibiting in this way pro-tein synthesis. AN plays an important role in the treatment of infections caused by members of the family Enterobacteriaceae and is often used in in-fections caused by organisms resistant to the other aminoglycosides. [10, 11].
Colistin (CL) is a cyclic cationic polypeptide belonging to polymyxin antibiotics. It disrupts the structure of the bacterial cell membrane by inter-acting with its phospholipids. It is one of the last resort antibiotics for multidrug resistant Gram-negative bacteria [12–14].
The objectives of this study were to examine: 1) the synergy of AN and NHS, 2) the synergy of CL and NHS 3) the synergy of the combination of AN and CL with NHS against colistin-resistant
C. freundii strains.
Material and Methods
Bacterial Strains
Two C. freundii strains isolated from the bron-choalveolar lavage of patients with respiratory tract infections hospitalized in the Lower Silesian J. Korczak Pediatric Centre, Wrocław, Poland were used.
Serum
Normal human serum (NHS) was obtained from healthy volunteers untreated with any an-timicrobial drug for the last three months. The
samples of NHS were collected, pooled and kept frozen in 0.5 mL portions at –70°C. The suitable volume of serum was thawed immediately before the experiment and used in a single experiment.
Antimicrobial Susceptibility
Testing
Minimal inhibitory concentration (MIC) as-says of two antimicrobial agents for C. freundii
strains were performed in Mueller-Hinton broth according to CLSI guidelines for broth microdilu-tion susceptibility testing [15].
Antimicrobial Agents
Amikacin (AN, Biodacyna®, Warsaw, Poland)
and colistin (CL, Colistin®, Tarchomin, Poland)
were used as delivered by producers.
Bactericidal Activity of NHS
The bactericidal activity of NHS was deter-mined as described previously [16]. Briefly, the strains were grown overnight, and then bacterial cells phase were transferred to a fresh nutrient broth (BIOMED, Warsaw, Poland) and incubated at 37°C for 30 min. Following incubation, the bac-terial cells were centrifuged (4000 rpm for 20 min). The bacteria suspended in 3.5 ml of phosphate-buffered saline (PBS) were then added to 50% NHS and to three mixtures of 50% NHS contain-ing ½ MIC of antimicrobial agents (NHS+AN, NHS+CL, NHS+AN+CL). Three different experi-mental controls were also used, including bacte-rial suspension and serum inactivated in 56°C for 30 min (HS56) with ½ MIC of antibiotics (HS56+AN, HS56+CL, HS56+AN+CL). All sam-ples were incubated in a water bath at 37°C. After 0, 60 and 180 min, samples were collected, diluted and cultured on nutrient agar plates (BIOMED, Warsaw, Poland) for 18 h at 37°C. The number of colony forming units (CFU) at time 0 was taken as 100%. Strains which showed a survival above 100% after 180 min of incubation in sera were re-garded as resistant.
Statistical Analysis
Results
In the first part of the experiments we deter-mined the susceptibility of the studied strains to 50% NHS. As seen in Tables 1 and 2, both strains were resistant to NHS and showed very strong survivability after 180 min of incubation. The per-centage of living bacterial cells was 3909.1% in the case of C. freundii 1338 and 1764.7% in the case of
C. freundii 1553.
In the next part of the experiments, the authors determined the MIC values of amikacin (AN) and colistin (CL). The mean values of the MIC were: AN – 0.25 µg/ml and CL – 8 µg/ml for C. freundii
1338; AN – 0.5 µg/ml and CL – 4 µg/ml for C. fre-undii 1553.
resistance to CL is currently rare and to date there is no clear explanation for this phenomenon. The Polish National reference Center for Micro-bial Susceptibility, in accordance with the data of The European Committee on Antimicrobial Susceptibility Testing (EUCAST), uses a cut off of 2 µg/ml or more as resistant. Both C. freundii
strains used in this research were considered to be colistin-resistant.
The characterization of the susceptibility of the strains to NHS and the determination of MIC values was followed by experiments in which an
Table 1. The survival of Citrobacter freundii 1338 strain in 50% NHS and antibiotics at ½ MICs
Tabela 1. Przeżywalność szczepu Citrobacter freundii 1338 w 50% NSL i ½ MIC antybiotyków
Serum and anti-biotics combina-tions (Surowica i kombinacje antybiotyków)
Time of incubation – min (Czas inkubacji – min)
0 60 180
CFUa CFU %b ± SD CFU % ± SD
NHS 1.1·107 1.5·108 1363.6 ± 347.4 4.3·108 3909.1 ± 1041.6
HS56 + AN 8.7·106 5.7·107 655.1 ± 286.1 1.4·108 1609.2 ± 523.3
NHS + AN 1.7·107 6.7·107 394.1 ± 137.3 3.2·107 188.2 ± 14.8
HS56 + CL 1.9·107 1.0·108 526.3 ± 156,4 4.6·108 2421.1 ± 93.4
NHS + CL 2.3·107 7.9·107 321.7 ± 143.2 2.9·106 12.6 ± 4.6
HS56 + AN + CL 2.7·107 1.2·108 444.4 ± 183.0 6.3·108 2333.3 ± 419.0
NHS + AN + CL 1.3·107 7.4·107 607.7 ± 210.5 1.2·107 92.3 ± 11.8 a CFU – colony forming units.
b % – percentage survival; (also relevant to Table 2). a CFU – jednostki tworzące kolonię.
b % – procent przeżywalności; (odnosi się również do tabeli 2).
Table 2. The survival of Citrobacter freundii 1553 strain in 50% NHS and antibiotics at ½ MICs
Tabela 2. Przeżywalność szczepu Citrobacter freundii 1553 w 50% NSL i ½ MIC antybiotyków
Serum and anti-biotics combina-tions (Surowica i kombinacje antybiotyków)
Time of incubation – min (Czas inkubacji – min)
0 60 180
CFU CFU % ± SD CFU % ± SD
NHS 1.7·107 4.4·107 253.5 ± 99.7 3.0·108 1764.7 ± 573.9
HS56 + AN 1.8·107 7.3·107 405.6 ± 177.9 6.7·108 3722.2 ± 889.3
NHS + AN 2.1·107 4.3·107 204.8 ± 88.6 5.8·107 276.1 ± 139.3
HS56 + CL 1.4·107 4.1·107 292.9 ± 112.3 3.0·108 2142.9 ± 937.2
NHS + CL 1.9·107 1.5·107 78.9 ± 19.2 1.1·107 64.7 ± 13.7
HS56 + AN + CL 2.3·107 1.1·108 478.3 ± 147.5 4.5·108 1956.5 ± 326.8
attempt was made to determine the synergistic ef-fect of NHS and ½ MIC of antimicrobial agents against bacteria.
The data in Table 1 show the growth inhibi-tion of C. freundii 1338 after exposure to NHS and ½ MIC of CL in comparison with C. freundii 1338 incubated in NHS. The survival percentage after 180 min of incubation was only 12.6%. This result is statistically significant (p = 0.0002)
As seen in Table 2, a synergistic inhibitory ef-fect between NHS and ½ MIC of CL against C. fre-undii 1553 was also observed. The percentage of surviving bacteria of C. freundii 1553 was 64.7% after 180 min of incubation (p = 0.0007).
Analyzing the results in Tables 1 and 2, when NHS was combined with AN, the bactericidal ac-tivity of NHS was enhanced against both C. freun-dii strains. The survival of bacteria treated with the AN+NHS mixture decreased 20.8 times for C. fre-undii 1338 (p = 0.0003) and 6.4 times in the case of
C. freundii 1553 (p = 0.002) after a 180-min incu-bation. Despite this, bacilli still remained resistant because the percentage of survival was higher than 100%.
The combination of NHS with AN and CL sub-MIC doses demonstrated strong bactericidal action against C. freundii 1553 bacilli (see Ta-ble 2). Bacteria were sensitive to a NHS+AN+CL mixture. The percentage of living cells was 1.2% after 180-min incubation. Bacterial growth was al-most 147 times reduced in comparison to bacteria treated with NHS, and this result was found to be statistically significant (p = 0.0007).
It is interesting that in the case of the second examined strain, the growth inhibition was much weaker. The survival percentage of the C. freundii
1338 strain was 92.3% after 180 min of incubation in the NHS+AN+CL mixture. Bacteria prolifer-ated 42.4 times less than in the mixture of NHS. Despite this, the results obtained were statistically significant (p = 0.0003).
As shown in Tables 1 and 2, the growth of C. freundii strains incubated in the presence of NHS and both antibiotics at subinhibitory concentra-tions was remarkably reduced compared to sus-pensions containing thermally inactivated serum and antimicrobial agents (p < 0.05). This result demonstrates the involvement of complement proteins in the bacterial cells lysis.
Discussion
Bacterial strains belonging to the Enterobacte-riaceae family are very often resistant to the bac-tericidal activity of human serum [5, 16, 17]. This phenomenon is rare within the Citrobacter genus,
however cases of serum-resistant bacilli have still been found [17]. The susceptibility to the serum’s bactericidal effect can be enhanced by addition of single or combined antibiotics [7–9].
Due to the increasing number of severe micro-bial infections and the more frequent drug resis-tance of bacterial strains, treatment with one an-tibiotic is often doomed to failure. Therefore, the use of antibiotic combinations becomes essential to effective treatment.
The experiments described in this paper were designed to elucidate the synergism between NHS and single (amikacin or colistin) or combined (amikacin and colistin) antibiotics. They were car-ried out with serum- and colistin-resistant C. fre-undii strains isolated from the bronchoalveolar lavage of children with pulmonary infection. Sig-nificant differences in the serum susceptibility of
C. freundii strains were observed after exposure of the bacteria to ½ MIC of colistin, whereas ½ MIC of amikacin did not alter the resistance of investi-gated rods to the bactericidal action of serum. In contrast to current results, Wojnicz and Cisowska [18]observed that amikacin at sub-lethal concen-trations affects the serum sensitivity of Escherichia coli strains.
The antimicrobial agents used in combina-tion with colistin are chosen empirically, without testing antibiotic synergism in vitro,and include many different groups, aminoglycosides e.g. ami-kacin, among others [12, 19].
The results of current study suggests a favor-able synergistic effect between amikacin and colis-tin, and their interaction with NHS. This combi-nation showed more effective bactericidal activity against C. freundii 1553 strain compared to the action of single antibiotics and NHS. The higher killing activity of combined colistin and amikacin is most likely caused by damage to the bacterial outer membrane by colistin and the facilitated ac-cess of amikacin into the cell.
Tascini et al. [20] also showed the synergistic activity of the combination of colistin with one or even two antibiotics against Enterobacter cloacae. A significant synergistic effect on serum bacteri-cidal activity was observed with the combination of amikacin and trovafloxacin against Pseudomonas aeruginosa and C. freundii by Lubash et al. [21].
proper-ties of the bacterial cells’ structures. Such a result was also obtained by Lubasch et al. [21]. They in-dicated that the bactericidal activity of cefepime alone was higher than that of the combination with trovafloxacin.
In conclusion, the interaction of serum and antibiotics is an important clinical issue and de-serves further study to clarify the mechanisms be-tween these two factors.
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Address for correspondence:
Dorota Tichaczek-Goska
Department of Biology and Medical Parasitology Wroclaw Medical University
Mikulicza-radeckiego 9 50-367 Wrocław, Poland Tel.: +48 71 7841523
E-mail: [email protected]
Conflict of interest: None declared
