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Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico
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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico b ˜ Paola Bocanenegra-Ibarias a , Cynthia Pena-López , Adrian Camacho-Ortiz c , d e Jorge Llaca-Díaz , Jesús Silva-Sánchez , Humberto Barrios e , Ulises Garza-Ramos e , Adrian Marcelo Rodríguez-Flores b , Elvira Garza-González a,d,∗ a
Servicio de Gastroenterología, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico Departamento de Medicina Interna, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico c Servicio de Infectología, Coordinación de Epidemiología Hospitalaria, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico d Departamento de Patología Clínica, Hospital Universitario Dr José Eleuterio González, Universidad Autónoma de Nuevo León, Nuevo León, Mexico e Instituto Nacional de Salud Pública, Departamento de Diagnóstico Epidemiológico, Cuernavaca, Morelos, Mexico b
a r t i c l e
i n f o
Article history: Received 12 May 2014 Accepted 27 October 2014 Keywords: Acinetobacter PFGE Carbapenemases OXA-58 OXA-72 Biofilm
a b s t r a c t The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of Acinetobacter baumannii clinical isolates obtained from a tertiary-care hospital in Mexico. Clinical isolates of A. baumannii (n = 152) isolated from 2007 to 2012 were included. Clonal diversity was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined using the broth microdilution method. IMP, VIM, NDM and OXA-type genes were screened by PCR. Biofilm production was analysed using the crystal violet method. Mortality attributable to A. baumannii infection was 14.5%. Fifty-four clones were detected, of which five predominated. MLST results showed three new sequence types and two reported sequence types. More than 86% of the isolates were resistant to ciprofloxacin, ceftazidime and cefotaxime. Furthermore, 50.7% and 35.5% of the isolates were resistant to imipenem and meropenem, respectively. Of the isolates evaluated, 28.3% and 25.7% were positive for the blaOXA-58 and blaOXA-72 genes, respectively. Biofilm production was associated with resistance to imipenem (P = 0.002). © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
2. Materials and methods
Carbapenem resistance in Acinetobacter baumannii is associated with the production of class B and class D -lactamases. Class B -lactamases are metallo--lactamases and class D lactamases are of the OXA-type [1]. The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of clinical isolates of A. baumannii obtained during a 6-year period in a tertiary-care university hospital in Mexico.
2.1. Clinical and demographic characteristics
∗ Corresponding author. Present address: Av. Madero s/n, Colonia Mitras Centro, Edificio Barragán, Segundo Piso, Monterrey, Nuevo León, C.P. 64460, Mexico. Tel.: +52 81 83 33 36 64; fax: +52 81 83 33 36 64. E-mail address: elvira garza [email protected] (E. Garza-González).
Demographic characteristics and the primary diagnosis were registered. Length of hospital stay (LOS) before and after isolation of A. baumannii, the hospital area where the clinical sample was taken, as well as patient risk factors for the presence of micro-organisms with multidrug resistance were recorded. The Charlson co-morbidity index and Acute Physiology and Chronic Health Evaluation (APACHE) II score were determined. Previous use of antibiotics was defined as administration of at least a single dose of an extended-spectrum antibiotic during the 3 weeks prior to the isolation of A. baumannii. Total mortality and attributable mortality were determined. The local ethics committee of the School of Medicine of the Universidad Autónoma de Nuevo León (Nuevo León, México) approved the study.
http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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2.2. Clinical specimens and wards
2.7. Biofilm production
Clinical isolates were obtained between 2007 and 2012 from the Dr José Eleuterio González University Hospital, a tertiarycare teaching hospital in Monterrey, Mexico. Most of the isolates were obtained from the respiratory tract (bronchial lavage and bronchoalveolar lavage; 64.5%), followed by blood (17.1%), cerebrospinal fluid (CSF) (5.3%), pleural fluid (3.9%), abscess (3.9%), abdominal biopsy (3.3%) and peritoneal fluid (2.0%). Isolates were collected predominantly from patients hospitalised in the intensive care unit (78.3%) as well as those in surgical wards (11.2%), the internal medicine department (7.2%) and other medical wards (3.3%).
Semi-quantitative determination of biofilm formation was performed by crystal violet staining as previously described [3]. The biofilm index [ratio of optical density at 595 nm to the optical density at 600 nm (OD595 /OD600 )] was used to normalise the amount of biofilm formed to the total cell content of each sample tested (OD600 ). The quality control organisms used were S. aureus ATCC 29213 (a high biofilm producer) and S. aureus ATCC BAA-44 (a low biofilm producer).
2.3. Identification of clinical isolates Acinetobacter baumannii complex identification was performed using Sensititre panels (TREK Diagnostic Systems Inc., Cleveland, OH) as described by the manufacturer. Identification of the A. baumannii species was made by sequence analysis of the recA gene and the 16S–23S rRNA intergenic spacer (ITS) [2].
2.8. Statistical analysis Statistical analysis was performed using one-way analysis of variance with Sidak post-hoc evaluation for comparison between multiple groups. The 2 or Fisher’s exact test was performed as needed. For correlation analysis, Spearman’s rank correlation was utilised. Values are reported as the mean ± standard deviation. A P-value of <0.05 was considered statistically significant. 3. Results 3.1. Demographic characteristics and clinical data
2.4. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis Chromosomal DNA was prepared using the suggested methodology for Staphylococcus aureus with modifications recommended for A. baumannii [3]. In five selected clinical isolates, MSLT was performed as described [4] and was analysed using the A. baumannii MLST website (http://pubmlst.org).
2.5. Antimicrobial susceptibility testing Antimicrobial susceptibility was determined using the broth microdilution method according to the 2013 Clinical and Laboratory Standards Institute (CLSI) guidelines in document M100-S23. The breakpoint for tigecycline was >2 mg/L as indicated by the British Society for Antimicrobial Chemotherapy (BSAC). Multidrug resistance was defined as having no susceptibility to at least one agent in three or more of the following classes of antimicrobials: fluoroquinolones; aminoglycosides; cephalosporins; carbapenems; polymyxins; and tetracycline [5].
Of the 152 patients included in the study, 77.6% were male. The mean age was 45 ± 17.1 years, LOS before isolation of A. baumannii was 16.18 ± 20.01 days and LOS after isolation of A. baumannii was 21.27 ± 26.14 days. The need for surgical intervention was the most frequent cause of hospitalisation, occurring in 62.5% of patients. On the day the sample was collected, patients had a mean Charlson comorbidity index of 1 ± 2.2 and a mean APACHE II score of 16 ± 11.7. The majority of patients had urinary catheters (92.1%), central lines (84.9%) and invasive mechanical ventilation (78.9%), whilst some patients had undergone abdominal surgical procedures (32.9%), had no abdominal procedures (44.1%) and were receiving parenteral nutrition (7.2%). The total mortality rate of the patients was 35.5% and the mortality rate attributable to A. baumannii infection was 14.5%. Before A. baumannii infection, 42.8% (65/152) of the patients received one or more doses of a second- or third-generation cephalosporin, 32.2% received carbapenems, 33.6% received vancomycin, 26.3% received quinolones and 3.9% received all of the aforementioned antibiotics. 3.2. Clonal diversity
2.6. Characterisation of carbapenemase genes PCR detection for IMP, NDM, VIM and OXA-type (blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like ) genes was performed using previously reported primers and conditions [6–9]. The positive OXA-24 and OXA-58 isolates were amplified in a second PCR using the primers OXA-24-F (5 -ATGAAAAAATTTATACTTCC-3 ), OXA-24-R (5 -TTAAATGATTCCAAGATTTTCTAG-3 ) and OXA-58R (5 -ATAAATAATGAAAAACACCCAAC-3 ) designed in this study as well as primer OXA-58-F previously reported [9]. All positive PCR products of the second PCR were sequenced using the chain termination method with a BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and were analysed on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Nucleotide sequences were compared with the GenBank database. The OXA-24-like and OXA-58-like genes flanked by the XerC/XerD recombination site and ISAba3 insertion sequences were determined by PCR using the primer XerC-R (5 ATTTCGCATAAGGCGTATTATGTTAATT-3 ) designed in this study.
Clones were detected by PFGE in 149 isolates and showed 54 different restriction patterns. Isolates that showed 100% similarity in their restriction pattern were classified as a clone. Of the 54 different clones obtained, 5 were classified as predominant clones as there were more than five isolates of these clones. Predominant clones were classified as clone A (24.2%), clone B (9.4%), clone C (8.7%), clone D (6.0%) and clone E (4.0%). Clone A was detected from 2007–2009. In 2009, it was displaced as the predominant clone by clone B, which was detected until 2011. Clone C was first detected in 2011 and remained present until the end of the study in 2012. Clones D and E were only detected in 2007. The presence of clone D was associated with previous use of third-generation cephalosporins (P = 0.001), and patients with clone A had a significantly higher LOS with a mean of 26.8 days (P = 0.0423) and a higher attributable mortality rate (P = 0.0008) compared with other clones. MLST analysis of the five selected isolates corresponded as follows: clone A isolate 3207, ST763; clone B isolate 3222, ST777; clone C isolate 3215, ST369; clone D isolate 3206, ST762; and clone E isolate 3224, ST229 (Table 1).
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Table 1 Characteristics of predominant clones of Acinetobacter baumannii. PFGE clone (n)
MLST of the isolate selected a
Origin of isolates (n)
Period of isolation
Hospital ward (n)
OXA-58
OXA-58/ISAba3
OXA-72
OXA-72/XerC
A (36)
ST763
−
−
+(1/14)
+(1/1)
+(6/14)
+(6/6)
C (13)
ST369
ICU (25), S (5), IM (5), O (1) ICU (12), IM (1), O (1) ICU (8), S (5)
+(13/30)
ST777
Feb. 2007–Aug. 2009 July 2009–March 2011 July 2011–June 2012
+(30/36)
B (14)
RT (28), B (2), CSF (2), O (4) RT (9), B (5)
−
−
+(12/13)
+(11/12)
D (9)
ST762
ICU (6), S (2), IM (1)
−
−
−
−
E (6)
ST229
Jan. 2007–July 2007 March 2007–July 2007
ICU (6)
−
−
−
−
RT (4), B (5), CSF (1), P (1), A (1), O (1) RT (6), CSF (1), P (1), O (1) RT (5), O (1)
PFGE, pulsed-field gel electrophoresis; MLST, multilocus sequence typing; RT, respiratory tract; B, blood; CSF, cerebrospinal fluid; O, others; P, pleural fluid; A, abscess; ICU, intensive care unit; S, surgical ward; IM, internal medicine. a One isolate of each clone was selected by MLST analysis. Clinical isolate corresponding to each clonal group: 3207 (A); 3222 (B); 3215 (C); 3206 (D); and 3224 (E).
3.3. Antimicrobial susceptibility and detection of carbapenemase genes
of blaOXA-58 was associated only with resistance to imipenem (P < 0.001) (Table 2).
Resistance levels were 89.5% for ceftazidime, 88.8% for ciprofloxacin, 86.8% for cefotaxime, 67.8% for tetracycline, 50.7% for amikacin and imipenem, 49.3% for gentamicin, 35.5% for meropenem, 23.7% for levofloxacin and 0.66% for colistin; no isolates were resistant to tigecycline. From 2007 to 2010 there was a decrease in the percentages of isolates resistant to ciprofloxacin, ceftazidime, cefotaxime, gentamicin and tetracycline (Fig. 1). In this study, 88.2% of the isolates were multidrug-resistant. All isolates tested positive for the blaOXA-51-like gene. Furthermore, 25.7% were positive for the blaOXA-24-like gene and by sequencing were confirmed as blaOXA-72 . Moreover, 28.3% of isolates were positive for the blaOXA-58 gene confirmed by sequencing. All isolates were negative for blaOXA-23-like , NDM, IMP and VIM genes. In addition, 46.5% of blaOXA-58- positive isolates were flanked by ISAba3 and 71.8% of blaOXA-72 -positive isolates were flanked by XerC. The presence of blaOXA-72 was associated with resistance to imipenem and meropenem (P < 0.0001). In contrast, the presence
3.4. Biofilm production There was no difference in the mean biofilm index between clones: clone B was the highest biofilm producer with a mean biofilm index of 2.18 ± 1.51; clone A had a mean biofilm index of 1.47 ± 1.70; clone C had a mean biofilm index of 1.45 ± 1.31; clone D had a mean biofilm index of 0.55 ± 0.53; and the lowest biofilm producer was clone E with a mean biofilm index of 0.51 ± 0.23. The mean biofilm index was 2.02 ± 0.53 for isolates obtained from the respiratory tract, 1.81 ± 1.87 from blood, 1.58 ± 0.99 from CSF, 1.46 ± 0.95 from pleural fluid, 1.34 ± 1.66 from peritoneal fluid, 1.28 ± 0.53 from an abdominal biopsy and 0.64 ± 0.32 from an abscess. Biofilm production was determined to be associated with high resistance to imipenem (P = 0.002). 4. Discussion Acinetobacter baumannii is a significant worldwide opportunistic pathogen that presents high drug resistance, environmental
Fig. 1. Percentage of isolates resistant to various antimicrobials. The percentage of isolates resistant to each antimicrobial tested changed during the course of the 6-year study. Data were broken down into 2-year intervals. CIP, ciprofloxacin; LVX, levofloxacin; GEN, gentamicin; AMK, amikacin; CAZ, ceftazidime; CTX, cefotaxime; IPM, imipenem; MEM, meropenem; CST, colistin; TET, tetracycline. No isolates were resistant to tigecycline.
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Table 2 Susceptibility profiles of carbapenem-resistant Acinetobacter baumannii isolates according to OXA production. Antibiotic
IPM MEM
blaOXA-58 positive (n = 43)
blaOXA-58 negative (n = 109)
MIC50
MIC90
n (%) R
MIC50
MIC90
n (%) R
32 8
32 16
38 (88.4) 15 (34.9)
2 <1
>64 >64
39 (35.8) 39 (35.8)
P-value
<0.0001 0.9172
blaOXA-72 positive (n = 39)
blaOXA-72 negative (n = 113)
MIC50
MIC90
n (%) R
MIC50
MIC90
n (%) R
>64 >64
>64 >64
39 (100) 39 (100)
2 <1
32 16
38 (33.6) 15 (13.3)
P-value
<0.0001 <0.0001
MIC50/90 , minimum inhibitory concentration (in mg/L) inhibiting 50% and 90% of the isolates, respectively; R, resistant; IPM, imipenem; MEM, meropenem.
persistence and causes nosocomial outbreaks of infection. In Mexico there are no reports of clonal diversity, antimicrobial resistance, detection of carbapenemase genes and biofilm production in A. baumannii. Therefore, it was important to analyse the behaviour of this bacterial species in a tertiary-care hospital in Monterrey, one of the most populated cities in Mexico. Risk factors for A. baumannii infection found in this study agree with those previously reported for this micro-organism, including prolonged hospitalisation, use of artificial devices, previous antimicrobial therapy and the presence of multiple co-morbid conditions. The mortality attributable to A. baumannii infection in analysed isolates was 14.5%, which is similar to the value of 7.8–23% previously reported by Falagas et al. [10]. The percentages of isolates resistant to ciprofloxacin, ceftazidime and gentamicin were similar to those reported in Latin America by the SENTRY Antimicrobial Surveillance Program in 2012 [11]. Furthermore, the reported resistance rates of 67.8% and 66.1% for imipenem and meropenem, respectively, in the SENTRY study [11] are lower than those obtained in the last 2 years of the present study, in which it was found that resistance to both imipenem and meropenem increased to 71.2%. During the course of the study, a decrease in the number of fluoroquinolones prescribed and an increase in the consumption of carbapenems was noted, which could influence the changes in the susceptibility patterns. These results confirmed the higher activity of levofloxacin than ciprofloxacin described previously [12]. Resistance of A. baumannii to fluoroquinolones has been related to mutations in the gyrA or parC genes that lead to changes in the structure of DNA gyrase or topoisomerase IV. It has been reported that in isolates with mutation in these genes, levofloxacin is up to three times more active than ciprofloxacin against A. baumannii strains with a single mutation and up to five times more active against strains with a double mutation [13]. These differences may be associated with mutations in gyrA or parC. There was no association between biofilm formation and the type of specimen or clone or the resistance profile. These results are different to those previously reported by Sanchez et al. [14], who found a correlation between biofilm formation and the site of isolate collection. Moreover, Sanchez et al. reported that strains capable of biofilm formation presented resistance to aminoglycosides, carbapenems, tetracycline and sulfonamides, whilst in the present study biofilm production was found to be associated with imipenem resistance. In Dr José Eleuterio González University Hospital, carbapenemresistant A. baumannii isolates with blaOXA-51 , blaOXA-58 and blaOXA-72 genes were detected. In Europe, the blaOXA-58 gene is commonly found, whilst the blaOXA-72 gene has been isolated sporadically. blaOXA-58 and blaOXA-72 have also been reported in the Western hemisphere; blaOXA-72 was found in 2011 in the USA and Brazil, whilst blaOXA-58 has been reported in Bolivia, Brazil and Argentina [1,15]. Thus, this study is the first report in Mexico and both genes were identified. The presence of Xer-like binding sites in association with blaOXA-72 in these isolates could be the mechanism responsible for the mobilisation of blaOXA-72 and this finding may explain, at least in part, the high frequency of this gene and its presence in different clones. Also, the insertion sequence ISAba3
could have mediated overexpression of blaOXA-58 as was previously described [1,15]. Of the 54 clones identified, 5 clones were considered predominant. Clones D and E were detected in only 1 year (2007) and these clones presented the lowest biofilm production and did not have blaOXA-58 or blaOXA-72 . The absence of biofilm production and antibiotic resistance-associated genes may have contributed to the low persistence of both of these clones. Furthermore, clones A, B and C presented similar biofilm production, thereby encouraging the persistence of these three clones. However, the main factor that may have favoured the displacement of clones across this 6-year study could have been the presence of OXA-type genes in the clones. Clone A was positive for blaOXA-58 , whereas clone B was positive for blaOXA-58 and blaOXA-72 and clone C was positive for blaOXA-72 only. The presence of blaOXA-58 was associated only with imipenem resistance, whilst the presence of blaOXA-72 was associated with meropenem and imipenem resistance. MLST results showed three new sequence types (ST762, ST763 and ST777) and two sequence types (ST229 and ST369) already deposited in the PubMLST database (http://pubmlst.org). However, only ST229 is part of the international clone CC222/228/229, which was previously described in Brazil and Japan [1]. In this 6-year study, an increase in meropenem resistance was observed because the A. baumannii strain that harbours blaOXA-58 was displaced by the A. baumannii clone that harbours blaOXA-72 . Replacement of blaOXA-58 by other OXA-type genes has been previously reported by Minandri et al., who found that blaOXA-23 displaced the pre-existing blaOXA-58 [15]. 5. Conclusion The prevalence of three clones was observed during the 6 years of study associated with biofilm production and the presence of OXA genes. A high resistance to several drugs analysed including imipenem was detected. A high frequency of blaOXA-58 and blaOXA-72 was detected. This is the first report of the presence of these genes in Mexico and of blaOXA-72 in Latin America. Acknowledgments The authors thank Lucy Acevedo and Carlos Paz for their assistance in the laboratory. Funding: This work was supported by grants from CONACyT (Mexican Council for Science and Technology) [CB-2009-01130224, 136339 and 169867]. Competing interests: None declared. Ethical approval: The local ethics committee of the School of Medicine of the Universidad Autónoma de Nuevo León (Nuevo León, México) approved this study [GA13-004]. References [1] Karah N, Sundsfjord A, Towner K, Samuelsen O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat 2012;15:237–47. [2] Chen TL, Siu LK, Wu RC, Shaio MF, Huang LY, Fung CP, et al. Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS)
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico b ˜ Paola Bocanenegra-Ibarias a , Cynthia Pena-López , Adrian Camacho-Ortiz c , d e Jorge Llaca-Díaz , Jesús Silva-Sánchez , Humberto Barrios e , Ulises Garza-Ramos e , Adrian Marcelo Rodríguez-Flores b , Elvira Garza-González a,d,∗ a
Servicio de Gastroenterología, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico Departamento de Medicina Interna, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico c Servicio de Infectología, Coordinación de Epidemiología Hospitalaria, Hospital Universitario Dr José Eleuterio González, Nuevo León, Mexico d Departamento de Patología Clínica, Hospital Universitario Dr José Eleuterio González, Universidad Autónoma de Nuevo León, Nuevo León, Mexico e Instituto Nacional de Salud Pública, Departamento de Diagnóstico Epidemiológico, Cuernavaca, Morelos, Mexico b
a r t i c l e
i n f o
Article history: Received 12 May 2014 Accepted 27 October 2014 Keywords: Acinetobacter PFGE Carbapenemases OXA-58 OXA-72 Biofilm
a b s t r a c t The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of Acinetobacter baumannii clinical isolates obtained from a tertiary-care hospital in Mexico. Clinical isolates of A. baumannii (n = 152) isolated from 2007 to 2012 were included. Clonal diversity was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined using the broth microdilution method. IMP, VIM, NDM and OXA-type genes were screened by PCR. Biofilm production was analysed using the crystal violet method. Mortality attributable to A. baumannii infection was 14.5%. Fifty-four clones were detected, of which five predominated. MLST results showed three new sequence types and two reported sequence types. More than 86% of the isolates were resistant to ciprofloxacin, ceftazidime and cefotaxime. Furthermore, 50.7% and 35.5% of the isolates were resistant to imipenem and meropenem, respectively. Of the isolates evaluated, 28.3% and 25.7% were positive for the blaOXA-58 and blaOXA-72 genes, respectively. Biofilm production was associated with resistance to imipenem (P = 0.002). © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
2. Materials and methods
Carbapenem resistance in Acinetobacter baumannii is associated with the production of class B and class D -lactamases. Class B -lactamases are metallo--lactamases and class D lactamases are of the OXA-type [1]. The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of clinical isolates of A. baumannii obtained during a 6-year period in a tertiary-care university hospital in Mexico.
2.1. Clinical and demographic characteristics
∗ Corresponding author. Present address: Av. Madero s/n, Colonia Mitras Centro, Edificio Barragán, Segundo Piso, Monterrey, Nuevo León, C.P. 64460, Mexico. Tel.: +52 81 83 33 36 64; fax: +52 81 83 33 36 64. E-mail address: elvira garza [email protected] (E. Garza-González).
Demographic characteristics and the primary diagnosis were registered. Length of hospital stay (LOS) before and after isolation of A. baumannii, the hospital area where the clinical sample was taken, as well as patient risk factors for the presence of micro-organisms with multidrug resistance were recorded. The Charlson co-morbidity index and Acute Physiology and Chronic Health Evaluation (APACHE) II score were determined. Previous use of antibiotics was defined as administration of at least a single dose of an extended-spectrum antibiotic during the 3 weeks prior to the isolation of A. baumannii. Total mortality and attributable mortality were determined. The local ethics committee of the School of Medicine of the Universidad Autónoma de Nuevo León (Nuevo León, México) approved the study.
http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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2.2. Clinical specimens and wards
2.7. Biofilm production
Clinical isolates were obtained between 2007 and 2012 from the Dr José Eleuterio González University Hospital, a tertiarycare teaching hospital in Monterrey, Mexico. Most of the isolates were obtained from the respiratory tract (bronchial lavage and bronchoalveolar lavage; 64.5%), followed by blood (17.1%), cerebrospinal fluid (CSF) (5.3%), pleural fluid (3.9%), abscess (3.9%), abdominal biopsy (3.3%) and peritoneal fluid (2.0%). Isolates were collected predominantly from patients hospitalised in the intensive care unit (78.3%) as well as those in surgical wards (11.2%), the internal medicine department (7.2%) and other medical wards (3.3%).
Semi-quantitative determination of biofilm formation was performed by crystal violet staining as previously described [3]. The biofilm index [ratio of optical density at 595 nm to the optical density at 600 nm (OD595 /OD600 )] was used to normalise the amount of biofilm formed to the total cell content of each sample tested (OD600 ). The quality control organisms used were S. aureus ATCC 29213 (a high biofilm producer) and S. aureus ATCC BAA-44 (a low biofilm producer).
2.3. Identification of clinical isolates Acinetobacter baumannii complex identification was performed using Sensititre panels (TREK Diagnostic Systems Inc., Cleveland, OH) as described by the manufacturer. Identification of the A. baumannii species was made by sequence analysis of the recA gene and the 16S–23S rRNA intergenic spacer (ITS) [2].
2.8. Statistical analysis Statistical analysis was performed using one-way analysis of variance with Sidak post-hoc evaluation for comparison between multiple groups. The 2 or Fisher’s exact test was performed as needed. For correlation analysis, Spearman’s rank correlation was utilised. Values are reported as the mean ± standard deviation. A P-value of <0.05 was considered statistically significant. 3. Results 3.1. Demographic characteristics and clinical data
2.4. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis Chromosomal DNA was prepared using the suggested methodology for Staphylococcus aureus with modifications recommended for A. baumannii [3]. In five selected clinical isolates, MSLT was performed as described [4] and was analysed using the A. baumannii MLST website (http://pubmlst.org).
2.5. Antimicrobial susceptibility testing Antimicrobial susceptibility was determined using the broth microdilution method according to the 2013 Clinical and Laboratory Standards Institute (CLSI) guidelines in document M100-S23. The breakpoint for tigecycline was >2 mg/L as indicated by the British Society for Antimicrobial Chemotherapy (BSAC). Multidrug resistance was defined as having no susceptibility to at least one agent in three or more of the following classes of antimicrobials: fluoroquinolones; aminoglycosides; cephalosporins; carbapenems; polymyxins; and tetracycline [5].
Of the 152 patients included in the study, 77.6% were male. The mean age was 45 ± 17.1 years, LOS before isolation of A. baumannii was 16.18 ± 20.01 days and LOS after isolation of A. baumannii was 21.27 ± 26.14 days. The need for surgical intervention was the most frequent cause of hospitalisation, occurring in 62.5% of patients. On the day the sample was collected, patients had a mean Charlson comorbidity index of 1 ± 2.2 and a mean APACHE II score of 16 ± 11.7. The majority of patients had urinary catheters (92.1%), central lines (84.9%) and invasive mechanical ventilation (78.9%), whilst some patients had undergone abdominal surgical procedures (32.9%), had no abdominal procedures (44.1%) and were receiving parenteral nutrition (7.2%). The total mortality rate of the patients was 35.5% and the mortality rate attributable to A. baumannii infection was 14.5%. Before A. baumannii infection, 42.8% (65/152) of the patients received one or more doses of a second- or third-generation cephalosporin, 32.2% received carbapenems, 33.6% received vancomycin, 26.3% received quinolones and 3.9% received all of the aforementioned antibiotics. 3.2. Clonal diversity
2.6. Characterisation of carbapenemase genes PCR detection for IMP, NDM, VIM and OXA-type (blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like ) genes was performed using previously reported primers and conditions [6–9]. The positive OXA-24 and OXA-58 isolates were amplified in a second PCR using the primers OXA-24-F (5 -ATGAAAAAATTTATACTTCC-3 ), OXA-24-R (5 -TTAAATGATTCCAAGATTTTCTAG-3 ) and OXA-58R (5 -ATAAATAATGAAAAACACCCAAC-3 ) designed in this study as well as primer OXA-58-F previously reported [9]. All positive PCR products of the second PCR were sequenced using the chain termination method with a BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and were analysed on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Nucleotide sequences were compared with the GenBank database. The OXA-24-like and OXA-58-like genes flanked by the XerC/XerD recombination site and ISAba3 insertion sequences were determined by PCR using the primer XerC-R (5 ATTTCGCATAAGGCGTATTATGTTAATT-3 ) designed in this study.
Clones were detected by PFGE in 149 isolates and showed 54 different restriction patterns. Isolates that showed 100% similarity in their restriction pattern were classified as a clone. Of the 54 different clones obtained, 5 were classified as predominant clones as there were more than five isolates of these clones. Predominant clones were classified as clone A (24.2%), clone B (9.4%), clone C (8.7%), clone D (6.0%) and clone E (4.0%). Clone A was detected from 2007–2009. In 2009, it was displaced as the predominant clone by clone B, which was detected until 2011. Clone C was first detected in 2011 and remained present until the end of the study in 2012. Clones D and E were only detected in 2007. The presence of clone D was associated with previous use of third-generation cephalosporins (P = 0.001), and patients with clone A had a significantly higher LOS with a mean of 26.8 days (P = 0.0423) and a higher attributable mortality rate (P = 0.0008) compared with other clones. MLST analysis of the five selected isolates corresponded as follows: clone A isolate 3207, ST763; clone B isolate 3222, ST777; clone C isolate 3215, ST369; clone D isolate 3206, ST762; and clone E isolate 3224, ST229 (Table 1).
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Table 1 Characteristics of predominant clones of Acinetobacter baumannii. PFGE clone (n)
MLST of the isolate selected a
Origin of isolates (n)
Period of isolation
Hospital ward (n)
OXA-58
OXA-58/ISAba3
OXA-72
OXA-72/XerC
A (36)
ST763
−
−
+(1/14)
+(1/1)
+(6/14)
+(6/6)
C (13)
ST369
ICU (25), S (5), IM (5), O (1) ICU (12), IM (1), O (1) ICU (8), S (5)
+(13/30)
ST777
Feb. 2007–Aug. 2009 July 2009–March 2011 July 2011–June 2012
+(30/36)
B (14)
RT (28), B (2), CSF (2), O (4) RT (9), B (5)
−
−
+(12/13)
+(11/12)
D (9)
ST762
ICU (6), S (2), IM (1)
−
−
−
−
E (6)
ST229
Jan. 2007–July 2007 March 2007–July 2007
ICU (6)
−
−
−
−
RT (4), B (5), CSF (1), P (1), A (1), O (1) RT (6), CSF (1), P (1), O (1) RT (5), O (1)
PFGE, pulsed-field gel electrophoresis; MLST, multilocus sequence typing; RT, respiratory tract; B, blood; CSF, cerebrospinal fluid; O, others; P, pleural fluid; A, abscess; ICU, intensive care unit; S, surgical ward; IM, internal medicine. a One isolate of each clone was selected by MLST analysis. Clinical isolate corresponding to each clonal group: 3207 (A); 3222 (B); 3215 (C); 3206 (D); and 3224 (E).
3.3. Antimicrobial susceptibility and detection of carbapenemase genes
of blaOXA-58 was associated only with resistance to imipenem (P < 0.001) (Table 2).
Resistance levels were 89.5% for ceftazidime, 88.8% for ciprofloxacin, 86.8% for cefotaxime, 67.8% for tetracycline, 50.7% for amikacin and imipenem, 49.3% for gentamicin, 35.5% for meropenem, 23.7% for levofloxacin and 0.66% for colistin; no isolates were resistant to tigecycline. From 2007 to 2010 there was a decrease in the percentages of isolates resistant to ciprofloxacin, ceftazidime, cefotaxime, gentamicin and tetracycline (Fig. 1). In this study, 88.2% of the isolates were multidrug-resistant. All isolates tested positive for the blaOXA-51-like gene. Furthermore, 25.7% were positive for the blaOXA-24-like gene and by sequencing were confirmed as blaOXA-72 . Moreover, 28.3% of isolates were positive for the blaOXA-58 gene confirmed by sequencing. All isolates were negative for blaOXA-23-like , NDM, IMP and VIM genes. In addition, 46.5% of blaOXA-58- positive isolates were flanked by ISAba3 and 71.8% of blaOXA-72 -positive isolates were flanked by XerC. The presence of blaOXA-72 was associated with resistance to imipenem and meropenem (P < 0.0001). In contrast, the presence
3.4. Biofilm production There was no difference in the mean biofilm index between clones: clone B was the highest biofilm producer with a mean biofilm index of 2.18 ± 1.51; clone A had a mean biofilm index of 1.47 ± 1.70; clone C had a mean biofilm index of 1.45 ± 1.31; clone D had a mean biofilm index of 0.55 ± 0.53; and the lowest biofilm producer was clone E with a mean biofilm index of 0.51 ± 0.23. The mean biofilm index was 2.02 ± 0.53 for isolates obtained from the respiratory tract, 1.81 ± 1.87 from blood, 1.58 ± 0.99 from CSF, 1.46 ± 0.95 from pleural fluid, 1.34 ± 1.66 from peritoneal fluid, 1.28 ± 0.53 from an abdominal biopsy and 0.64 ± 0.32 from an abscess. Biofilm production was determined to be associated with high resistance to imipenem (P = 0.002). 4. Discussion Acinetobacter baumannii is a significant worldwide opportunistic pathogen that presents high drug resistance, environmental
Fig. 1. Percentage of isolates resistant to various antimicrobials. The percentage of isolates resistant to each antimicrobial tested changed during the course of the 6-year study. Data were broken down into 2-year intervals. CIP, ciprofloxacin; LVX, levofloxacin; GEN, gentamicin; AMK, amikacin; CAZ, ceftazidime; CTX, cefotaxime; IPM, imipenem; MEM, meropenem; CST, colistin; TET, tetracycline. No isolates were resistant to tigecycline.
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Table 2 Susceptibility profiles of carbapenem-resistant Acinetobacter baumannii isolates according to OXA production. Antibiotic
IPM MEM
blaOXA-58 positive (n = 43)
blaOXA-58 negative (n = 109)
MIC50
MIC90
n (%) R
MIC50
MIC90
n (%) R
32 8
32 16
38 (88.4) 15 (34.9)
2 <1
>64 >64
39 (35.8) 39 (35.8)
P-value
<0.0001 0.9172
blaOXA-72 positive (n = 39)
blaOXA-72 negative (n = 113)
MIC50
MIC90
n (%) R
MIC50
MIC90
n (%) R
>64 >64
>64 >64
39 (100) 39 (100)
2 <1
32 16
38 (33.6) 15 (13.3)
P-value
<0.0001 <0.0001
MIC50/90 , minimum inhibitory concentration (in mg/L) inhibiting 50% and 90% of the isolates, respectively; R, resistant; IPM, imipenem; MEM, meropenem.
persistence and causes nosocomial outbreaks of infection. In Mexico there are no reports of clonal diversity, antimicrobial resistance, detection of carbapenemase genes and biofilm production in A. baumannii. Therefore, it was important to analyse the behaviour of this bacterial species in a tertiary-care hospital in Monterrey, one of the most populated cities in Mexico. Risk factors for A. baumannii infection found in this study agree with those previously reported for this micro-organism, including prolonged hospitalisation, use of artificial devices, previous antimicrobial therapy and the presence of multiple co-morbid conditions. The mortality attributable to A. baumannii infection in analysed isolates was 14.5%, which is similar to the value of 7.8–23% previously reported by Falagas et al. [10]. The percentages of isolates resistant to ciprofloxacin, ceftazidime and gentamicin were similar to those reported in Latin America by the SENTRY Antimicrobial Surveillance Program in 2012 [11]. Furthermore, the reported resistance rates of 67.8% and 66.1% for imipenem and meropenem, respectively, in the SENTRY study [11] are lower than those obtained in the last 2 years of the present study, in which it was found that resistance to both imipenem and meropenem increased to 71.2%. During the course of the study, a decrease in the number of fluoroquinolones prescribed and an increase in the consumption of carbapenems was noted, which could influence the changes in the susceptibility patterns. These results confirmed the higher activity of levofloxacin than ciprofloxacin described previously [12]. Resistance of A. baumannii to fluoroquinolones has been related to mutations in the gyrA or parC genes that lead to changes in the structure of DNA gyrase or topoisomerase IV. It has been reported that in isolates with mutation in these genes, levofloxacin is up to three times more active than ciprofloxacin against A. baumannii strains with a single mutation and up to five times more active against strains with a double mutation [13]. These differences may be associated with mutations in gyrA or parC. There was no association between biofilm formation and the type of specimen or clone or the resistance profile. These results are different to those previously reported by Sanchez et al. [14], who found a correlation between biofilm formation and the site of isolate collection. Moreover, Sanchez et al. reported that strains capable of biofilm formation presented resistance to aminoglycosides, carbapenems, tetracycline and sulfonamides, whilst in the present study biofilm production was found to be associated with imipenem resistance. In Dr José Eleuterio González University Hospital, carbapenemresistant A. baumannii isolates with blaOXA-51 , blaOXA-58 and blaOXA-72 genes were detected. In Europe, the blaOXA-58 gene is commonly found, whilst the blaOXA-72 gene has been isolated sporadically. blaOXA-58 and blaOXA-72 have also been reported in the Western hemisphere; blaOXA-72 was found in 2011 in the USA and Brazil, whilst blaOXA-58 has been reported in Bolivia, Brazil and Argentina [1,15]. Thus, this study is the first report in Mexico and both genes were identified. The presence of Xer-like binding sites in association with blaOXA-72 in these isolates could be the mechanism responsible for the mobilisation of blaOXA-72 and this finding may explain, at least in part, the high frequency of this gene and its presence in different clones. Also, the insertion sequence ISAba3
could have mediated overexpression of blaOXA-58 as was previously described [1,15]. Of the 54 clones identified, 5 clones were considered predominant. Clones D and E were detected in only 1 year (2007) and these clones presented the lowest biofilm production and did not have blaOXA-58 or blaOXA-72 . The absence of biofilm production and antibiotic resistance-associated genes may have contributed to the low persistence of both of these clones. Furthermore, clones A, B and C presented similar biofilm production, thereby encouraging the persistence of these three clones. However, the main factor that may have favoured the displacement of clones across this 6-year study could have been the presence of OXA-type genes in the clones. Clone A was positive for blaOXA-58 , whereas clone B was positive for blaOXA-58 and blaOXA-72 and clone C was positive for blaOXA-72 only. The presence of blaOXA-58 was associated only with imipenem resistance, whilst the presence of blaOXA-72 was associated with meropenem and imipenem resistance. MLST results showed three new sequence types (ST762, ST763 and ST777) and two sequence types (ST229 and ST369) already deposited in the PubMLST database (http://pubmlst.org). However, only ST229 is part of the international clone CC222/228/229, which was previously described in Brazil and Japan [1]. In this 6-year study, an increase in meropenem resistance was observed because the A. baumannii strain that harbours blaOXA-58 was displaced by the A. baumannii clone that harbours blaOXA-72 . Replacement of blaOXA-58 by other OXA-type genes has been previously reported by Minandri et al., who found that blaOXA-23 displaced the pre-existing blaOXA-58 [15]. 5. Conclusion The prevalence of three clones was observed during the 6 years of study associated with biofilm production and the presence of OXA genes. A high resistance to several drugs analysed including imipenem was detected. A high frequency of blaOXA-58 and blaOXA-72 was detected. This is the first report of the presence of these genes in Mexico and of blaOXA-72 in Latin America. Acknowledgments The authors thank Lucy Acevedo and Carlos Paz for their assistance in the laboratory. Funding: This work was supported by grants from CONACyT (Mexican Council for Science and Technology) [CB-2009-01130224, 136339 and 169867]. Competing interests: None declared. Ethical approval: The local ethics committee of the School of Medicine of the Universidad Autónoma de Nuevo León (Nuevo León, México) approved this study [GA13-004]. References [1] Karah N, Sundsfjord A, Towner K, Samuelsen O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat 2012;15:237–47. [2] Chen TL, Siu LK, Wu RC, Shaio MF, Huang LY, Fung CP, et al. Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS)
Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022
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Please cite this article in press as: Bocanenegra-Ibarias P, et al. Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.10.022