Antimicrobial Section / Original Paper Received: January 22, 2015 Accepted: August 24, 2015 Published online: November 5, 2015
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Biofilm Formation and Susceptibility to Polymyxin B by a Highly Prevalent Clone of MultidrugResistant Acinetobacter baumannii from a Mexican Tertiary Care Hospital Roberto Rosales-Reyes a María Dolores Alcántar-Curiel a Ma. Dolores Jarillo-Quijada a Catalina Gayosso-Vázquez a María del Rayo Morfin-Otero b Eduardo Rodríguez-Noriega b José Ignacio Santos-Preciado a
a Unidad
de Investigación en Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, and b Hospital Civil de Guadalajara, Fray Antonio Alcalde, Universidad de Guadalajara, Guadalajara, Mexico
Abstract Background: Acinetobacter baumannii has emerged as a major cause of hospital-associated infections with increased morbidity and mortality among those affected. Methods: A total of 85 isolates of a highly prevalent multidrug-resistant clone, identified during the period 2007–2011, were analyzed for biofilm formation on a polystyrene surface. The minimal inhibitory concentration was determined by the Sensititre System, the agar disk diffusion method and then read by means of the BIOMIC system and serial dilutions on Müller-Hinton agar. Results: In this study, covering a period of 5 years (2007–2011), we demonstrate that a particular clone emerged as the most prevalent, with an associated lethality of 28.2%. We demonstrate that 92.9% of strains corresponding to this clone are biofilm producers. Our results also demonstrate that all isolates were 100% susceptible to polymyxin B. Conclusion: Our study suggests that the high prevalence and lethality of this multidrug-resistant clone of
© 2015 S. Karger AG, Basel 0009–3157/15/0611–0008$39.50/0 E-Mail [email protected] www.karger.com/che
A. baumannii and its persistence over close to 5 years in a Mexican tertiary hospital environment can be explained in part by the ability of these clinical isolates of A. baumannii to form biofilms. © 2015 S. Karger AG, Basel
Introduction
Acinetobacter baumannii is a Gram-negative, aerobic, nonmotile coccobacillus that belongs to a genus comprised of both environmental bacterial species and opportunistic pathogens. These bacteria have been isolated from different environmental sources, including soil, water and food products, and are often isolated from medical devices [1–4]. The formation of biofilm, which consists of groups of bacterial cells adhering to a surface and being enclosed within a self-produced extracellular matrix to mediate their protection from different adverse en-
R. Rosales-Reyes and M.D. Alcántar-Curiel contributed equally to this work.
Roberto Rosales-Reyes Unidad de Investigación en Medicina Experimental Facultad de Medicina, Universidad Nacional Autónoma de México Av. Dr. Balmis 148, Colonia Doctores, Mexico D.F. (Mexico) E-Mail robros @ yahoo.com
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Key Words Acinetobacter baumannii · Multidrug resistance · Biofilm · Polymyxin B
belonged to clone-22. All isolates were identified using API20NE (bioMérieux® SA) [13]. The identification of A. baumannii was confirmed by determining the presence of the bla-OXA-51-like gene by PCR [14, 15]. Briefly, the amplification was performed using the forward and reverse primers (FW: ATGAACATTMAARCRCTCTTACTTA and RV: CTATAAAATACCTAATTMTTCTAA). For each PCR reaction, we used 1 μmol of each primer in 1× master mix green (GoTaq® Promega) and a 100-ng DNA template for amplification reactions. Samples were heated to 94 ° C for 5 min, 35 cycles of 94 ° C for 1 min, 50 ° C for 1 min and 72 ° C for 1.5 min, with a final extension step at 72 ° C for 15 min. Amplification reactions were carried out using an iCycler (BioRad®) thermocycler and the resulting products were separated by electrophoresis in 1% agarose gels at 120 V, using a Power Station 300 chamber (Labnet International, Inc.). Gels were stained with ethidium bromide for 3 min to visualize the separated bands. Amplified products were subjected to nucleotide sequencing at the Instituto de Biotecnología, Universidad Nacional Autónoma de México [14]. Only 1 isolate per patient was studied. All isolates used in this study belonged to the A. baumannii-calcoaceticus complex. Nosocomial infections were defined according to the criteria published by the Centers for Disease Control and Prevention and by physicians from the Infectious Diseases Unit [16].
Biofilm Production The biofilm production was quantified as previously described [17]. Briefly, 5 ml of overnight cultures grown at 37 ° C were diluted to OD600 0.003 in LB media, and triplicate 500-μl aliquots were dispensed into polystyrene tubes. Following 24 h of static incubation at 37 ° C, the medium was removed and the tubes were washed gently once with deionized water. Adherent bacteria were stained with 1% (w/v) crystal violet and washed 3 times with deionized water. The bound crystal violet was dissolved in 1 ml of 100% methanol and quantified by measuring OD540 nm.
Bacterial Collection and Microbiological Methods In this study, we analyzed 2,405 isolates of A. baumannii collected between January 2007 and December 2011. Only 303 (12.6%) were associated with nosocomial infections and 85 of these
Antimicrobial Susceptibility Testing The minimum inhibitory concentration (MIC) was determined using broth microdilution applied to Sensititre plates (TREK Diagnostics Systems, Inc., Westlake, Ohio, USA). The antimicrobial agents tested include amikacin, gentamicin, tobramycin, ceftazidime, cefotaxime, cefepime, ciprofloxacin, levofloxacin and trimethoprim-sulfamethoxazole. The MIC to imipenem and meropenem was performed by agar disk diffusion [18] and the reading results with the BIOMIC system (Giles Scientific, Santa Barbara, Calif., USA). The BIOMIC V3 microbiology system provides an automated interpretative reading of the antibiotic disk diffusion (Kirby-Bauer) test on plates, based on the expert review of the Clinical and Laboratory Standards Institute (CLSI) guidelines, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the British Society for Antimicrobial Chemotherapy (BSAC). The MIC to PB was determined by serial dilution on agar [18]. Briefly, the strains were grown on MacConkey agar the day before the experiment. The 0.5 McFarland standard and a 1:10 dilution were used to adjust the bacterial inoculum density. At the same time, a stock solution of 1,280 μg/ml of PB (Sigma-Aldrich) was immediately prepared and diluted 1: 2 to get a solution of 2.5 μg/ml of PB. Each solution was diluted 1: 10 on Müller-Hinton (MH) agar to get final concentrations of 128–0.25 μg/ml. The plates (MH agar with PB) were inoculated using a
Characterization of a Multidrug-Resistant Clone of A. baumannii
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Material and Methods
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vironmental conditions, is an important feature of most clinical isolates of Acinetobacter spp. [5, 6]. A. baumannii has emerged as an important nosocomial and opportunistic pathogen that causes serious, often life-threatening infections in health care facilities worldwide. The emergence of clinical isolates with resistance to multiple antibiotics [7–9] greatly limits treatment options, especially if the organism is resistant to the carbapenem class of antibiotics [10]. In a previous report [11], we identified an increase in the isolation of multidrug-resistant A. baumannii in different wards of the Hospital Civil de Guadalajara (HCG-Mexico), a 1,000-bed, tertiary referral teaching hospital in western Mexico, with variable resistance to diverse antibiotics, including carbapenems. However, susceptibility to polymyxin B (PB) was not tested. In the same study, we identified 13 clones by pulsefield gel electrophoresis from 65 isolates; the most prevalent clone exhibited 100% resistance to meropenem and 90.1% to imipenem [11]. During a period extending from 2007 to 2011, we identified the persistence of a particular clone (clone-22), which was associated with 85 episodes of nosocomial infections and 1 hospital outbreak, which lasted 6 months and accounted for 35 separate nosocomial infections and 4 deaths. The prevalence of A. baumannii clone-22 during almost 5 years (the study period) suggested that the isolates of this clone could have factors that confer an advantage for its colonization and for biofilm production. Although the correlation between antibiotic resistance and biofilm production has already been reported for A. baumannii [7], we raised the question as to whether, in addition to the multidrug resistance exhibited by this clone, its production of biofilm could also account for its persistence in the hospital environment. To test this hypothesis, we measured the biofilm production and antimicrobial susceptibility in the 85 clinical isolates. Although the literature has shown that A. baumannii is a bacterium with a high resistance to multiple antibiotics, it seems to maintain a high susceptibility to polymyxins [12]. In our previous study, the rate of PB resistance was not tested for any of the isolates including clone-22. In this study, we evaluated the susceptibility to PB of the 85 clinical isolates corresponding to clone-22 by means of agar dilution and in vitro susceptibility techniques.
Steers replicator with pins of 3 mm in diameter to deliver approximately 2 μl with 1 × 104 colony-forming units (CFUs) onto the agar surface. The plates were dried and incubated during 18 h at 37 ° C. The MIC was determined as the lowest concentration of PB in which the A. baumannii growth was inhibited. The classification of each isolate as susceptible or resistant was established according to the CLSI [18]. Escherichia coli strain ATCC 25922 was employed for quality control tests during the susceptibility assays.
Table 1. Distribution of A. baumannii isolates by hospital ward
Ward
Isolates, n
Gastroenterology Hematology/Oncology Infectious Diseases Intensive Care Unit Intensive Care Unit (Neonatology) Internal Medicine Nephrology Neurosurgery Orthopedic Oncology Pediatric Plastic Surgery Proctology Surgery Thorax/Cardiology Traumatology
4 4 11 14 1 11 5 12 2 1 3 3 1 5 1 7
Total
85
Total, %
Killing Assay To determine the efficacy of PB in the reduction of bacterial load, we used an in vitro killing assay described for Pseudomonas aeruginosa with modifications [19]. Briefly, from an overnight culture, we adjusted the inoculum to 1 × 107 bacteria in PBS, at the same time as preparing MH broth and MH broth plus 1 or 10 μg/ ml of fresh PB. The samples were incubated for 3 h at 37 ° C without shaking. After the incubation, the bacterial cultures were diluted 1:10, and 20 μl of each dilution was plated on MH agar. The agar plates were dried and incubated for 18 h at 37 ° C. CFUs were determined and multiplied by the dilution. The susceptibility to PB was determined in relation to the bacterial CFUs obtained in PBS.
4.71 4.71 12.94 16.47 1.18 12.94 5.88 14.12 2.35 1.18 3.53 3.53 1.18 5.88 1.18 8.24 100
Results are shown as the number of isolates by ward and the percentage.
Results
Antibiotic Susceptibility of A. baumannii Clone-22 The antimicrobial susceptibility results showed that clone-22 is highly resistant to 5 families of antibiotics (table 3). Susceptibility to fluoroquinolones was 1.2% to both ciprofloxacin and levofloxacin. Susceptibility to carbapenems was 5.9% to imipenem and 4.7% to meropenem. Susceptibility to aminoglycosides was 8.2% to amikacin, 4.7% to gentamicin and 5.9% to tobramycin. There was variable susceptibility to cephalosporins, i.e. 2.3% to ceftazidime, 0.0% to both cefotaxime and ceftriaxone and 12.9% to cefepime. The susceptibility to antibiotics related to the folate pathway, such as trimethoprim-sulfa10
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Table 2. Distribution of A. baumannii isolates according to their isolation site
Ward
Isolates, n (%)
Blood Respiratory sources Wound secretions Vascular catheter Urine
14 (16.47) 7 (8.24) 46 (54.12) 13 (15.29) 5 (5.88)
methoxazole, was 2.4%. In contrast, all isolates of clone-22 were 100% susceptible to PB (table 3). In order to demonstrate the efficacy of PB to decrease the number of CFUs of members of the A. baumannii clone-22, we performed a killing assay, taking into account that the MIC reported in the CLSI guidelines for A. baumannii is ≤2 μg/ml of PB [18]. Our results show that all A. baumannii clinical isolates were susceptible to ≤1 μg/ml of PB (table 3). In the assay, we incubated bacteria in PBS with 1 or 10 μg/ml of PB for 3 h at 37 ° C. The results show that 1 μg/ml of PB decreased the CFUs by 98.78 ± 1.22% of the initial load and that 10 μg/ml decreased it by 99.89 ± 0.11% (fig. 3).
Rosales-Reyes et al.
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A. baumannii Clone-22 Is Highly Efficient at Producing Biofilm Of the 2,405 isolates of A. baumannii collected between January 2007 and December 2011, only 303 (12.6%) were associated with nosocomial infections, and 85 of these belonged to clone-22. The distribution of these isolates according to hospital ward is shown in table 1 and according to their isolation site in table 2. In figure 1, we show the distribution of all A. baumannii isolates by hospital ward on the time line. The result shows that 79/85 (92.95%) of the clinical isolates were biofilm producers and only 6/85 (7.05%) were weak biofilm producers (fig. 2). In addition, 2 days of incubation did not significantly increase the biofilm formation (data not shown).
Fig. 1. Detection of A. baumannii clone-22. During a period of 5 years, we detected 85 unique clinical isolates.
The isolation according to hospital ward is presented.
Fig. 2. Biofilm formation by A. baumannii clone-22. Eighty-five clinical isolates were assessed with regard to the
Characterization of a Multidrug-Resistant Clone of A. baumannii
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
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formation of biofilm on polystyrene surfaces. We used the strain A. baumannii ATCC-19606. Values represent the mean ± standard error of the mean from 2 independent experiments done in triplicate. **** p ≤ 0.0001.
Table 3. MIC data and resistance of A. baumannii (n = 85)
Antibiotic family
Drug
Breakpoint [18], μg/ml
MIC50, MIC90, MIC range, μg/ml μg/ml μg/ml
Resistant
Susceptible
n
%
n
%
Aminoglucosidesa
AMK GEN TOB
S ≤16 S ≤4 S ≤4
R ≥64 R ≥16 R ≥16
32 ≥8 8
≥64 ≥64 ≥8
≤2 ≤1 ≤1
≥64 ≥64 ≥8
74 78 80
87.1 91.8 94.1
7 4 5
8.2 4.7 5.9
Cephemsa
CAZ CTX FEP CRO
S ≤8 S ≤8 S ≤8 S ≤8
R ≥32 R ≥64 R ≥32 R ≥64
16 32 16 32
≥32 ≥32 ≥32 ≥32
≤1 32 ≤2 32
≥32 ≥32 ≥32 ≥32
81 82 46 82
95.4 96.5 54.2 96.5
2 0 11 0
2.3 0 12.9 0
Fluoroquinolonesa
CIP LVX
S ≤1 S ≤2
R ≥4 R ≥8
2 4
≥4 ≥8
≤2 4
≥4 ≥8
84 83
98.8 97.6
1 1
1.2 1.2
Folate pathway inhibitorsa
SXT
S ≤2/38 R ≥4/76
≥4/76
82
97.6
2
2.4
Carbapenemsb
IPM MEM
S ≤2 S ≤2
R ≥8 R ≥8
8 8
≥24 ≥16
80 81
94.1 95.3
5 4
5.9 4.7
Lipopeptidesc
PB
S ≤2
R ≥4
0.5
2
0
0
85
≤2/38
≥4/76 ≤2/38 ≥24 ≥16 0.5
1 ≤2 ≤0.125
100
a MIC was performed by the Sensititre II method to: AMK = amikacin; CAZ = ceftazidime; CIP = ciprofloxacin; CRO = ceftriaxone; CTX = cefotaxime; FEP = cefepime; GEN = gentamycin; LVX = levofloxacin; SXT = trimethoprim-sulfamethoxazole; TOB = tobramycin. b The susceptibility test was performed by the agar disk diffusion method and the reading of MIC with the BIOMIC system: IPM = imipenem; MEM = meropenem. c MICs were performed by serial dilutions on MH agar and PB.
Fig. 3. A. baumannii load reduction by PB-killing assay. Eightyfive clinical isolates (clone-22) were assessed with PB-killing assay during 3 h. The CFUs were determined 24 h after treatment. On the y-axis, we show the killing percent of A. baumannii (susceptibility) after PB treatment. On the x-axis, we show the use of A. baumannii ATCC-19606 (as type strain) and the clinical isolates (clone-22) under PB treatment. Each value represents the mean of 1 experiment in triplicate.
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Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
A. baumannii has emerged as the second most important pathogen causing infections in a tertiary care teaching hospital in Guadalajara, Jalisco, Mexico [11]. A prevalent and persistent clone, which we classified as clone-22, was the most predominant A. baumannii isolate in the period 2007–2011. Our results showed a low frequency of distribution of this clone across several wards. The mortality rate associated with A. baumannii infection was 28.23%. In other countries, the mortality rate due to A. baumannii infections is high and ranges from 22 to 59% [20, 21]. Persistence of bacteria on abiotic surfaces is commonly associated with their ability to produce biofilms [22]. There are limited reported data concerning the ability of A. baumannii to form biofilms. Rodríguez-Baño et al. [23] found that 63% of 92 clonally unrelated A. baumannii clinical isolates form biofilm. Sechi et al. [24] found that 16/20 isolates (80%) were able to produce biofilms, suggesting that this high rate of biofilm production is due to a predominant clone. Similarly, we found that 79/85 (92.9%) clonally related A. baumannii strains were biofilm producers. Our data suggest that biofilm production Rosales-Reyes et al.
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Discussion
by bacterial communities is a clone-specific feature, and that it is possible that this expression is similar under different conditions found in HCG-Mexico and that biofilm production by A. baumannii clone-22 could indeed be a plausible explanation for the persistent prevalence in all wards of the HCG-Mexico. The ability to form biofilms is associated to multidrug resistance and, specifically, with the expression of the blaPER-1 gene [7, 25]. In a recent study [14], we identified that 84% of A. baumannii isolates were resistant to carbapenems; this resistance is associated with the production of metallo-β-lactamases. In addition, 49.6 and 1.2% of these isolates carried the blaOXA-72 and blaVIM-1 genes, respectively. The spread of multidrug-resistant A. baumannii strains among critically ill, hospitalized patients and subsequent outbreaks have become an increasing cause for concern. Although members of clone-22 carried a reduced susceptibility to meropenem and imipenem (4.7 and 5.9%, respectively) [14], all members of this clone were susceptible to PB (100%).
Conclusions
Our results strongly suggest that the high prevalence and lethality of this multidrug-resistant clone of A. baumannii and its persistence for close to a 5-year period in the hospital environment could be explained by the ability of the clinical isolates of this clone to form biofilms. Additional studies are needed to test this correlation. Finally, although it was not the original hypothesis of this study, our finding that some isolates of this clone display susceptibility to PB justifies the clinical use of this antibiotic in the treatment of critically ill patients, as has been suggested by others [26]. It remains to be demonstrated if the use of PB will have any potential impact on the development of biofilms by this clone.
Acknowledgment This work was supported in part by UNAM-DGAPA-PAPIIT, grant No. IN220613.
References
Characterization of a Multidrug-Resistant Clone of A. baumannii
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Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Biofilm Formation and Susceptibility to Polymyxin B by a Highly Prevalent Clone of MultidrugResistant Acinetobacter baumannii from a Mexican Tertiary Care Hospital Roberto Rosales-Reyes a María Dolores Alcántar-Curiel a Ma. Dolores Jarillo-Quijada a Catalina Gayosso-Vázquez a María del Rayo Morfin-Otero b Eduardo Rodríguez-Noriega b José Ignacio Santos-Preciado a
a Unidad
de Investigación en Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, and b Hospital Civil de Guadalajara, Fray Antonio Alcalde, Universidad de Guadalajara, Guadalajara, Mexico
Abstract Background: Acinetobacter baumannii has emerged as a major cause of hospital-associated infections with increased morbidity and mortality among those affected. Methods: A total of 85 isolates of a highly prevalent multidrug-resistant clone, identified during the period 2007–2011, were analyzed for biofilm formation on a polystyrene surface. The minimal inhibitory concentration was determined by the Sensititre System, the agar disk diffusion method and then read by means of the BIOMIC system and serial dilutions on Müller-Hinton agar. Results: In this study, covering a period of 5 years (2007–2011), we demonstrate that a particular clone emerged as the most prevalent, with an associated lethality of 28.2%. We demonstrate that 92.9% of strains corresponding to this clone are biofilm producers. Our results also demonstrate that all isolates were 100% susceptible to polymyxin B. Conclusion: Our study suggests that the high prevalence and lethality of this multidrug-resistant clone of
© 2015 S. Karger AG, Basel 0009–3157/15/0611–0008$39.50/0 E-Mail [email protected] www.karger.com/che
A. baumannii and its persistence over close to 5 years in a Mexican tertiary hospital environment can be explained in part by the ability of these clinical isolates of A. baumannii to form biofilms. © 2015 S. Karger AG, Basel
Introduction
Acinetobacter baumannii is a Gram-negative, aerobic, nonmotile coccobacillus that belongs to a genus comprised of both environmental bacterial species and opportunistic pathogens. These bacteria have been isolated from different environmental sources, including soil, water and food products, and are often isolated from medical devices [1–4]. The formation of biofilm, which consists of groups of bacterial cells adhering to a surface and being enclosed within a self-produced extracellular matrix to mediate their protection from different adverse en-
R. Rosales-Reyes and M.D. Alcántar-Curiel contributed equally to this work.
Roberto Rosales-Reyes Unidad de Investigación en Medicina Experimental Facultad de Medicina, Universidad Nacional Autónoma de México Av. Dr. Balmis 148, Colonia Doctores, Mexico D.F. (Mexico) E-Mail robros @ yahoo.com
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Key Words Acinetobacter baumannii · Multidrug resistance · Biofilm · Polymyxin B
belonged to clone-22. All isolates were identified using API20NE (bioMérieux® SA) [13]. The identification of A. baumannii was confirmed by determining the presence of the bla-OXA-51-like gene by PCR [14, 15]. Briefly, the amplification was performed using the forward and reverse primers (FW: ATGAACATTMAARCRCTCTTACTTA and RV: CTATAAAATACCTAATTMTTCTAA). For each PCR reaction, we used 1 μmol of each primer in 1× master mix green (GoTaq® Promega) and a 100-ng DNA template for amplification reactions. Samples were heated to 94 ° C for 5 min, 35 cycles of 94 ° C for 1 min, 50 ° C for 1 min and 72 ° C for 1.5 min, with a final extension step at 72 ° C for 15 min. Amplification reactions were carried out using an iCycler (BioRad®) thermocycler and the resulting products were separated by electrophoresis in 1% agarose gels at 120 V, using a Power Station 300 chamber (Labnet International, Inc.). Gels were stained with ethidium bromide for 3 min to visualize the separated bands. Amplified products were subjected to nucleotide sequencing at the Instituto de Biotecnología, Universidad Nacional Autónoma de México [14]. Only 1 isolate per patient was studied. All isolates used in this study belonged to the A. baumannii-calcoaceticus complex. Nosocomial infections were defined according to the criteria published by the Centers for Disease Control and Prevention and by physicians from the Infectious Diseases Unit [16].
Biofilm Production The biofilm production was quantified as previously described [17]. Briefly, 5 ml of overnight cultures grown at 37 ° C were diluted to OD600 0.003 in LB media, and triplicate 500-μl aliquots were dispensed into polystyrene tubes. Following 24 h of static incubation at 37 ° C, the medium was removed and the tubes were washed gently once with deionized water. Adherent bacteria were stained with 1% (w/v) crystal violet and washed 3 times with deionized water. The bound crystal violet was dissolved in 1 ml of 100% methanol and quantified by measuring OD540 nm.
Bacterial Collection and Microbiological Methods In this study, we analyzed 2,405 isolates of A. baumannii collected between January 2007 and December 2011. Only 303 (12.6%) were associated with nosocomial infections and 85 of these
Antimicrobial Susceptibility Testing The minimum inhibitory concentration (MIC) was determined using broth microdilution applied to Sensititre plates (TREK Diagnostics Systems, Inc., Westlake, Ohio, USA). The antimicrobial agents tested include amikacin, gentamicin, tobramycin, ceftazidime, cefotaxime, cefepime, ciprofloxacin, levofloxacin and trimethoprim-sulfamethoxazole. The MIC to imipenem and meropenem was performed by agar disk diffusion [18] and the reading results with the BIOMIC system (Giles Scientific, Santa Barbara, Calif., USA). The BIOMIC V3 microbiology system provides an automated interpretative reading of the antibiotic disk diffusion (Kirby-Bauer) test on plates, based on the expert review of the Clinical and Laboratory Standards Institute (CLSI) guidelines, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the British Society for Antimicrobial Chemotherapy (BSAC). The MIC to PB was determined by serial dilution on agar [18]. Briefly, the strains were grown on MacConkey agar the day before the experiment. The 0.5 McFarland standard and a 1:10 dilution were used to adjust the bacterial inoculum density. At the same time, a stock solution of 1,280 μg/ml of PB (Sigma-Aldrich) was immediately prepared and diluted 1: 2 to get a solution of 2.5 μg/ml of PB. Each solution was diluted 1: 10 on Müller-Hinton (MH) agar to get final concentrations of 128–0.25 μg/ml. The plates (MH agar with PB) were inoculated using a
Characterization of a Multidrug-Resistant Clone of A. baumannii
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Material and Methods
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vironmental conditions, is an important feature of most clinical isolates of Acinetobacter spp. [5, 6]. A. baumannii has emerged as an important nosocomial and opportunistic pathogen that causes serious, often life-threatening infections in health care facilities worldwide. The emergence of clinical isolates with resistance to multiple antibiotics [7–9] greatly limits treatment options, especially if the organism is resistant to the carbapenem class of antibiotics [10]. In a previous report [11], we identified an increase in the isolation of multidrug-resistant A. baumannii in different wards of the Hospital Civil de Guadalajara (HCG-Mexico), a 1,000-bed, tertiary referral teaching hospital in western Mexico, with variable resistance to diverse antibiotics, including carbapenems. However, susceptibility to polymyxin B (PB) was not tested. In the same study, we identified 13 clones by pulsefield gel electrophoresis from 65 isolates; the most prevalent clone exhibited 100% resistance to meropenem and 90.1% to imipenem [11]. During a period extending from 2007 to 2011, we identified the persistence of a particular clone (clone-22), which was associated with 85 episodes of nosocomial infections and 1 hospital outbreak, which lasted 6 months and accounted for 35 separate nosocomial infections and 4 deaths. The prevalence of A. baumannii clone-22 during almost 5 years (the study period) suggested that the isolates of this clone could have factors that confer an advantage for its colonization and for biofilm production. Although the correlation between antibiotic resistance and biofilm production has already been reported for A. baumannii [7], we raised the question as to whether, in addition to the multidrug resistance exhibited by this clone, its production of biofilm could also account for its persistence in the hospital environment. To test this hypothesis, we measured the biofilm production and antimicrobial susceptibility in the 85 clinical isolates. Although the literature has shown that A. baumannii is a bacterium with a high resistance to multiple antibiotics, it seems to maintain a high susceptibility to polymyxins [12]. In our previous study, the rate of PB resistance was not tested for any of the isolates including clone-22. In this study, we evaluated the susceptibility to PB of the 85 clinical isolates corresponding to clone-22 by means of agar dilution and in vitro susceptibility techniques.
Steers replicator with pins of 3 mm in diameter to deliver approximately 2 μl with 1 × 104 colony-forming units (CFUs) onto the agar surface. The plates were dried and incubated during 18 h at 37 ° C. The MIC was determined as the lowest concentration of PB in which the A. baumannii growth was inhibited. The classification of each isolate as susceptible or resistant was established according to the CLSI [18]. Escherichia coli strain ATCC 25922 was employed for quality control tests during the susceptibility assays.
Table 1. Distribution of A. baumannii isolates by hospital ward
Ward
Isolates, n
Gastroenterology Hematology/Oncology Infectious Diseases Intensive Care Unit Intensive Care Unit (Neonatology) Internal Medicine Nephrology Neurosurgery Orthopedic Oncology Pediatric Plastic Surgery Proctology Surgery Thorax/Cardiology Traumatology
4 4 11 14 1 11 5 12 2 1 3 3 1 5 1 7
Total
85
Total, %
Killing Assay To determine the efficacy of PB in the reduction of bacterial load, we used an in vitro killing assay described for Pseudomonas aeruginosa with modifications [19]. Briefly, from an overnight culture, we adjusted the inoculum to 1 × 107 bacteria in PBS, at the same time as preparing MH broth and MH broth plus 1 or 10 μg/ ml of fresh PB. The samples were incubated for 3 h at 37 ° C without shaking. After the incubation, the bacterial cultures were diluted 1:10, and 20 μl of each dilution was plated on MH agar. The agar plates were dried and incubated for 18 h at 37 ° C. CFUs were determined and multiplied by the dilution. The susceptibility to PB was determined in relation to the bacterial CFUs obtained in PBS.
4.71 4.71 12.94 16.47 1.18 12.94 5.88 14.12 2.35 1.18 3.53 3.53 1.18 5.88 1.18 8.24 100
Results are shown as the number of isolates by ward and the percentage.
Results
Antibiotic Susceptibility of A. baumannii Clone-22 The antimicrobial susceptibility results showed that clone-22 is highly resistant to 5 families of antibiotics (table 3). Susceptibility to fluoroquinolones was 1.2% to both ciprofloxacin and levofloxacin. Susceptibility to carbapenems was 5.9% to imipenem and 4.7% to meropenem. Susceptibility to aminoglycosides was 8.2% to amikacin, 4.7% to gentamicin and 5.9% to tobramycin. There was variable susceptibility to cephalosporins, i.e. 2.3% to ceftazidime, 0.0% to both cefotaxime and ceftriaxone and 12.9% to cefepime. The susceptibility to antibiotics related to the folate pathway, such as trimethoprim-sulfa10
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
Table 2. Distribution of A. baumannii isolates according to their isolation site
Ward
Isolates, n (%)
Blood Respiratory sources Wound secretions Vascular catheter Urine
14 (16.47) 7 (8.24) 46 (54.12) 13 (15.29) 5 (5.88)
methoxazole, was 2.4%. In contrast, all isolates of clone-22 were 100% susceptible to PB (table 3). In order to demonstrate the efficacy of PB to decrease the number of CFUs of members of the A. baumannii clone-22, we performed a killing assay, taking into account that the MIC reported in the CLSI guidelines for A. baumannii is ≤2 μg/ml of PB [18]. Our results show that all A. baumannii clinical isolates were susceptible to ≤1 μg/ml of PB (table 3). In the assay, we incubated bacteria in PBS with 1 or 10 μg/ml of PB for 3 h at 37 ° C. The results show that 1 μg/ml of PB decreased the CFUs by 98.78 ± 1.22% of the initial load and that 10 μg/ml decreased it by 99.89 ± 0.11% (fig. 3).
Rosales-Reyes et al.
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A. baumannii Clone-22 Is Highly Efficient at Producing Biofilm Of the 2,405 isolates of A. baumannii collected between January 2007 and December 2011, only 303 (12.6%) were associated with nosocomial infections, and 85 of these belonged to clone-22. The distribution of these isolates according to hospital ward is shown in table 1 and according to their isolation site in table 2. In figure 1, we show the distribution of all A. baumannii isolates by hospital ward on the time line. The result shows that 79/85 (92.95%) of the clinical isolates were biofilm producers and only 6/85 (7.05%) were weak biofilm producers (fig. 2). In addition, 2 days of incubation did not significantly increase the biofilm formation (data not shown).
Fig. 1. Detection of A. baumannii clone-22. During a period of 5 years, we detected 85 unique clinical isolates.
The isolation according to hospital ward is presented.
Fig. 2. Biofilm formation by A. baumannii clone-22. Eighty-five clinical isolates were assessed with regard to the
Characterization of a Multidrug-Resistant Clone of A. baumannii
Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
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formation of biofilm on polystyrene surfaces. We used the strain A. baumannii ATCC-19606. Values represent the mean ± standard error of the mean from 2 independent experiments done in triplicate. **** p ≤ 0.0001.
Table 3. MIC data and resistance of A. baumannii (n = 85)
Antibiotic family
Drug
Breakpoint [18], μg/ml
MIC50, MIC90, MIC range, μg/ml μg/ml μg/ml
Resistant
Susceptible
n
%
n
%
Aminoglucosidesa
AMK GEN TOB
S ≤16 S ≤4 S ≤4
R ≥64 R ≥16 R ≥16
32 ≥8 8
≥64 ≥64 ≥8
≤2 ≤1 ≤1
≥64 ≥64 ≥8
74 78 80
87.1 91.8 94.1
7 4 5
8.2 4.7 5.9
Cephemsa
CAZ CTX FEP CRO
S ≤8 S ≤8 S ≤8 S ≤8
R ≥32 R ≥64 R ≥32 R ≥64
16 32 16 32
≥32 ≥32 ≥32 ≥32
≤1 32 ≤2 32
≥32 ≥32 ≥32 ≥32
81 82 46 82
95.4 96.5 54.2 96.5
2 0 11 0
2.3 0 12.9 0
Fluoroquinolonesa
CIP LVX
S ≤1 S ≤2
R ≥4 R ≥8
2 4
≥4 ≥8
≤2 4
≥4 ≥8
84 83
98.8 97.6
1 1
1.2 1.2
Folate pathway inhibitorsa
SXT
S ≤2/38 R ≥4/76
≥4/76
82
97.6
2
2.4
Carbapenemsb
IPM MEM
S ≤2 S ≤2
R ≥8 R ≥8
8 8
≥24 ≥16
80 81
94.1 95.3
5 4
5.9 4.7
Lipopeptidesc
PB
S ≤2
R ≥4
0.5
2
0
0
85
≤2/38
≥4/76 ≤2/38 ≥24 ≥16 0.5
1 ≤2 ≤0.125
100
a MIC was performed by the Sensititre II method to: AMK = amikacin; CAZ = ceftazidime; CIP = ciprofloxacin; CRO = ceftriaxone; CTX = cefotaxime; FEP = cefepime; GEN = gentamycin; LVX = levofloxacin; SXT = trimethoprim-sulfamethoxazole; TOB = tobramycin. b The susceptibility test was performed by the agar disk diffusion method and the reading of MIC with the BIOMIC system: IPM = imipenem; MEM = meropenem. c MICs were performed by serial dilutions on MH agar and PB.
Fig. 3. A. baumannii load reduction by PB-killing assay. Eightyfive clinical isolates (clone-22) were assessed with PB-killing assay during 3 h. The CFUs were determined 24 h after treatment. On the y-axis, we show the killing percent of A. baumannii (susceptibility) after PB treatment. On the x-axis, we show the use of A. baumannii ATCC-19606 (as type strain) and the clinical isolates (clone-22) under PB treatment. Each value represents the mean of 1 experiment in triplicate.
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Chemotherapy 2015–2016;61:8–14 DOI: 10.1159/000440605
A. baumannii has emerged as the second most important pathogen causing infections in a tertiary care teaching hospital in Guadalajara, Jalisco, Mexico [11]. A prevalent and persistent clone, which we classified as clone-22, was the most predominant A. baumannii isolate in the period 2007–2011. Our results showed a low frequency of distribution of this clone across several wards. The mortality rate associated with A. baumannii infection was 28.23%. In other countries, the mortality rate due to A. baumannii infections is high and ranges from 22 to 59% [20, 21]. Persistence of bacteria on abiotic surfaces is commonly associated with their ability to produce biofilms [22]. There are limited reported data concerning the ability of A. baumannii to form biofilms. Rodríguez-Baño et al. [23] found that 63% of 92 clonally unrelated A. baumannii clinical isolates form biofilm. Sechi et al. [24] found that 16/20 isolates (80%) were able to produce biofilms, suggesting that this high rate of biofilm production is due to a predominant clone. Similarly, we found that 79/85 (92.9%) clonally related A. baumannii strains were biofilm producers. Our data suggest that biofilm production Rosales-Reyes et al.
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Discussion
by bacterial communities is a clone-specific feature, and that it is possible that this expression is similar under different conditions found in HCG-Mexico and that biofilm production by A. baumannii clone-22 could indeed be a plausible explanation for the persistent prevalence in all wards of the HCG-Mexico. The ability to form biofilms is associated to multidrug resistance and, specifically, with the expression of the blaPER-1 gene [7, 25]. In a recent study [14], we identified that 84% of A. baumannii isolates were resistant to carbapenems; this resistance is associated with the production of metallo-β-lactamases. In addition, 49.6 and 1.2% of these isolates carried the blaOXA-72 and blaVIM-1 genes, respectively. The spread of multidrug-resistant A. baumannii strains among critically ill, hospitalized patients and subsequent outbreaks have become an increasing cause for concern. Although members of clone-22 carried a reduced susceptibility to meropenem and imipenem (4.7 and 5.9%, respectively) [14], all members of this clone were susceptible to PB (100%).
Conclusions
Our results strongly suggest that the high prevalence and lethality of this multidrug-resistant clone of A. baumannii and its persistence for close to a 5-year period in the hospital environment could be explained by the ability of the clinical isolates of this clone to form biofilms. Additional studies are needed to test this correlation. Finally, although it was not the original hypothesis of this study, our finding that some isolates of this clone display susceptibility to PB justifies the clinical use of this antibiotic in the treatment of critically ill patients, as has been suggested by others [26]. It remains to be demonstrated if the use of PB will have any potential impact on the development of biofilms by this clone.
Acknowledgment This work was supported in part by UNAM-DGAPA-PAPIIT, grant No. IN220613.
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Characterization of a Multidrug-Resistant Clone of A. baumannii
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