CC BY-NC-ND
0Accepted Manuscript
Title: Molecular Epidemiology and Characterization of Multiple-Drug Resistant (MDR) Clinical Isolates of Acinetobacter baumannii
Author: Sherief El-Shazly Ali Dashti Leila Vali Michael Bolaris Ashraf S. Ibrahim
PII: DOI:
Reference:
S1201-9712(15)00250-7 http://dx.doi.org/doi:10.1016/j.ijid.2015.10.016 IJID 2461
To appear in: International Journal of Infectious Diseases
Received date: 26-3-2015
Revised date: 5-10-2015
Accepted date: 22-10-2015
Please cite this article as: El-Shazly S, Dashti A, Vali L, Bolaris M, Ibrahim AS, Molecular Epidemiology and Characterization of Multiple-Drug Resistant (MDR) Clinical Isolates of Acinetobacter baumannii, International Journal of Infectious Diseases (2015), http://dx.doi.org/10.1016/jijid.2015.10.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Highlights
3 • Oxacillinases correlated with 90% of MDR A. baumannii from a single hospital
5 • GES-type carbapenemases and ISAba1 were also detected.
7 • Insertion elements are likely behind resistance via P-lactamase production
9 • Majority of the strains belonged to sequence type-2 10
11 • Genotyping of resistance aids in understanding of MDR A. baumannii transmission
12 Molecular Epidemiology and Characterization of Multiple-Drug Resistant
13 (MDR) Clinical Isolates of Acinetobacter baumannii
14 Sherief El-Shazly1, 2, Ali Dashti1, Leila Vali1, Michael Bolaris3, Ashraf S. Ibrahim2, 4*
16 department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Health Sciences
17 Center, Kuwait University, Kuwait; 2Division of Adult Infectious Diseases, Los Angeles
18 Biomedical Research Institute at Harbor-University of California Los Angeles (UCLA) Medical
19 Center, Torrance, California, USA; 3Division of Pediatric Infectious Diseases, Los Angeles
20 Biomedical Research Institute at Harbor-University of California Los Angeles (UCLA) Medical
21 Center, Torrance, California, USA; 4David Geffen School of Medicine at UCLA, Los Angeles,
22 California, USA
25 Running Title: Molecular epidemiology of MDR A. baumannii
28 *Corresponding author: Ashraf S. Ibrahim, PhD, Los Angeles Biomedical Research Institute,
29 Division of Infectious Diseases, Harbor-UCLA Medical Center, 1124 West Carson St., St.
30 John's Cardiovascular Research Center, Torrance, CA 90502. Phone 310-222-6424, Fax 31031 782-2016; ibrahim@labiomed.org.
36 Abstract:
37 Objectives: We aimed to identify the genetic relatedness of multiple-drug resistance (MDR) in
38 Acinetobacter baumannii clinical isolates recovered from a hospital in Los Angeles.
39 Methods: Twenty one MDR A. baumannii isolates were collected and their antibiotic
40 susceptibility were determined according to the CLSI guidelines. Genes coding for antibiotic
41 resistance were identified by PCR and their identities were confirmed by DNA sequencing.
42 Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE) and multi-locus
43 sequence typing (MLST).
44 Results: MDR consistently correlated with the presence of oxacillinases, mostly in the form of
45 plasmid-mediated OXA-23 enzyme which were detected in 12 (57.1%) isolates. GES-type
46 carbapenemases were found in 20 (95.2%) strains, AAC in all 21 (100%) strains, PER in 7
47 (33.3%) strains and ISAba/ has been detected in 16 (76.2%) isolates. The association between
48 ISAba/ and resistant genes confirms insertion elements as a source of P-lactamase production.
49 Of the 21 clinical isolates, 5 were found to be related to sequence type-1 (ST1) and 16 to ST2 as
50 analyzed by MLST. PFGE demonstrated that the majority of clinical isolates are highly related
51 (>85%).
52 Conclusions: This study supports a more complete understanding of genotyping of antibiotic
53 resistance for better assessment of MDR strains transmission.
55 Keywords: A. baumannii, P-lactamase, MLST, PFGE
56 1. Background:
58 Acinetobacter baumannii has emerged as a predominant cause of healthcare-associated infections
59 (including those seen in wounded soldiers) both in the United States and world-wide.1-10 A.
60 baumannii infections include pneumonia (especially ventilator associated pneumonia), wound
61 infections, urinary tract infections, septicemia and surgical site infections.11-13 Risk factors for
62 A. baumannii infections, especially in elderly individuals, include; patients having underlying
63 diseases (e.g. diabetes), immune suppression, burns, trauma, invasive medical procedures,
64 mechanical ventilation, catheters, previous antibiotic treatments and extended hospital stay.14 Of
65 great concern is the recent rise in the frequency of multiple drug resistant (MDR) and extremely
66 drug resistant (XDR)- A. baumannii infections.2'11'12 The percentage of A. baumannii infections
67 caused by MDR strains (defined as resistance to >1 agent in at least three antimicrobial
68 categories) and XDR strains (defined as resistance to all available antibiotics except colistin and
69 tigecycline) has increased from <4% in 2000 to 60-70% in 2010.14 Infections caused by MDR A.
70 baumannii are associated with longer hospitalization, greater healthcare costs, greater morbidity,
71 and >60% mortality for bloodstream infections as compared to drug-susceptible strains.15-18
72 XDR infections are treatable only with second-line agents, such as tigecycline and colistin which
73 are associated with clinical failure, development of resistance and nephrotoxicity.16,19-28 Further,
74 pandrug-resistant infections (PDR) are resistant to every FDA approved antibiotic, and are hence
75 untreatable. Because of the difficulty in treating MDR, XDR and PDR A. baumannii infections,
76 surveillance of A. baumannii isolates represent the cornerstone of prevention and control of these
77 infections. In the current study, we aimed at characterizing the resistance mechanisms and
78 determining the genetic relatedness of clinical isolates recovered from Harbor-UCLA Medical
79 Center at Los Angeles County.
80 2. Methods:
82 2.1. Bacterial Identifications
83 Twenty four clinical strains of A. baumannii obtained from in-patients Harbor-UCLA Medical
84 Center (HUMC) of which 21 isolates were investigated and identified to the species level by
85 using Vitek2 (BioMerieux Vitek Systems Inc., USA) and MicroScan (WalkAway System,
86 Siemens, USA) systems utilizing biochemical methods.
88 2.2. Antimicrobial Susceptibility Testing
89 Antimicrobial susceptibility testing was determined for all HUMC strains of A. baumannii by
90 automated broth microdilution method (Vitek2) (Vitek AMS; BioMerieux Vitek Systems Inc.,
91 USA) and the results were analyzed and interpreted using clinical breakpoints according to the
92 Clinical and Laboratory Standards Institute (CLSI) guidelines.29 The antibiotics tested were:
93 Amikacin, amoxicillin/clavulanic acid, ampicillin/sulbactam, ampicillin, cefazolin, cefepime,
94 cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cefoxitin, cefpodoxime, cephalothin,
95 ceftriazone, ciprofloxacin, gentamicin, imipenem, meropenem, levofloxacin, nitrofurantoin,
96 norfloxacin, tetracycline, tobramycin, trimethoprim/sulfamethoxazole, piperacillin/tazobactam,
97 pipercillin, tigecycline, colistin and tigecycline. Extended spectrum P-lactamase (ESBL)
98 production was confirmed by Vitek2 analyzer, Microscan and disk diffusion tests. Minimum
99 inhibitory concentration (MICs) of quinolones, fluoroquinolones and P-lactams including
100 carbapenems were determined using the E-test method (CLSI 2012).29 Isolates that showed
101 resistance to at least three classes of antibiotics were considered as MDR strains, whereas
isolates showing resistance to all antibiotics except for colistin and tigecycline were considered as XDR strains.
2.3. Identification of Housekeeping Genes
Bacterial DNA was extracted using QIAquick PCR Purification Kit (Qiagen, USA). Primers used for polymerase chain reaction (PCR) amplifications of seven housekeeping genes in A. baumannii are listed in Table 1.
2.4. Antibiotic Resistance Genes by PCR and DNA Sequencing
The presence of resistant genes (Table 2) was investigated by PCR using GoTaq Green Master Mix (Promega, USA). PCR was conducted in a GeneAmp 9700 system (Perkin-Elmer, Illinois, USA) using the conditions specified for each primer as corresponding to the reference source. PCR was performed for metallo-P-lactamase and ESBL-encoding genes including bla SIM (Seoul imipenemase), bla VIM (Verona integron-encoded metallo- P -lactamases), bla VEB (Vietnamese extended-spectrum- P -lactamase) and bla IMP (Imipenemase), bla TEM-1 (Temoneira) and bla SHV (Sulfhydryl variable), bla CTX-M-like (Cefotaximase-Munchen),30 bla NDM-1 (New-Delhi metallo- P -lactamase),31 qnrA, qnrB and qnrS (Quinolone resistance genes),32 aac (N-acetyltransferase),33 gyrA (DNA gyrase subunit A) and parC (Topoisomerase IV subunit C),34 AmpC (class C P-lactamases),35 bla PER (Pseudomonas extended resistance),36 bla GES (Guiana extended spectrum),36 OXA-(oxacillinases)-encoding genes including bla OXA-51-like, bla OXA-58-like, bla OXA-48-like, bla OXA-23-like, bla OXA-24-like
37 38 39 40
genes, ' IS (insertion sequence), and ISAbal. Amplified PCR products were purified with Qiagen purification kit (Qiagen, USA) according to the manufacturer's instructions and both
strands were sequenced by automated AB13100 DNA sequencer (Applied BioSystems) system. The BLAST program of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov) was used to search and compare databases for similar nucleotide acid sequences.
2.5. Pulsed-Field Gel Electrophoresis
Pulsed-Field Gel Electrophoresis (PFGE) analysis was based on techniques described elsewhere.41 PFGE of ApaI-digested genomic DNA (Promega, UK) from each HUMC strain was performed to detect the relationships among the clinical isolates of A. baumannii. After PFGE, the gels were stained with ethidium bromide and scanned. The analysis of gels was performed using BioNumerics software version 7.1 (Applied Maths, Ghent, Belgium). This software facilitates the development of the algorithms necessary for the comparison of profiles of isolates based on the Dice coefficient and the hierarchic unweighted pair arithmetic average algorithm. Cluster analysis and phylogenetic trees were subsequently analyzed with an optimization of 1.0% and a tolerance of 0.7%. Isolates were considered to belong to the same PFGE clone if their Dice similarity index was >85%.
2.6. Multi Locus Sequence Typing
Multi-locus sequence typing (MLST) was based on a sequence analysis of the internal fragments of seven housekeeping genes: cpn60 (60-KDa chaperonin), fusA (elongation factor EF-G), gltA (citrate synthase), pyrG (CTP synthase), recA (homologous recombination factor), rplB (50S ribosomal protein L2), rpoB (RNA polymerase subunit B). The MLST scheme including amplification and sequencing primers, allele sequences and sequence types (STs) were available
at Institute Pasteur's MLST web site
(http://www.pasteur.fr/recherche/genopole/PF8/mlst/references_Abaumannii.html). The
housekeeping genes for the MLST scheme were selected on the basis of their sequence availability in GenBank and prior studies of the phylogenetic relationships for the genus Acinetobacter and their presence in other MLST schemes available for other bacterial species. PCR primers were chosen from previous studies or were designed for amplification of the seven selected genes (Table 1).
All PCR amplifications were carried out using GoTaq Green Matser Mix (Promega, USA) under the following conditions: 35 cycles (denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 30 sec) proceeded by a 2 min denaturation at 94°C and followed by a 5 min extension at 72°C. PCR products were directly purified from the reaction mixture with the QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's recommendations. Sequencing of internal DNA fragments of 297 bp to 633 bp of the selected housekeeping genes was performed using ABI Prism 377 sequencer using the ABI Prism BigDye terminator cycle sequencing ready reaction kit V3.1 (PE Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. PCR primers were used for sequencing on both strands. Sequence data were aligned by CLUSTALW (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
2.7. Plasmid Curing Experiments
Plasmid curing procedure was performed for five selected HUMC isolates (HUMC-1, HUMC-3, HUMC-4, HUMC-10, HUMC-19) of A. baumannii using 47oC as a growing temperature. The five isolates were selected to represent the presence or absence of resistance genes and ISAba1.
Plasmid curing using temperature as a curing agent were examined after 3 days incubation at 47C followed by antibiotic sensitivity testing.
3. Results:
3.1. Antimicrobial Susceptibilities to A. baumannii Isolates
A total of 21 clinical samples were analyzed in 2011. The sources of the isolates included 9 samples of respiratory secretions (42.9%), 4 samples of sputum (19%), 2 samples from abdominal secretions (9.5%), 2 samples from wounds (9.5%) and one sample each from other sites (4.8%) including urine, bronchoalveolar lavage, foot and groin samples. Antibiotic sensitivity testing revealed that all the isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, tetracycline, ticarcillin/K clavulanate, whereas two isolates were found to be sensitive to meropenem, eight to imipenem, three to amikacin, four to ampicillin/sulbactam, one to each of trimethoprim/sulfamethoxazole tobramycin. All clinical isolates of A. baumannii were sensitive to colistin, and tigecycline with the exception of one and two strains, respectively. Susceptibility testing results of the studied clinical isolates are summarized in Table 3.
3.2. Characterization of Carbapenemases and other p-Lactamase Genes
We identified the presence of oxacillinases, mostly in the form of plasmid-mediated OXA-23 enzyme which were detected in 12 (57.1%) isolates as well as P-lactamase resistant genes in 7 isolates harboring PER (33.3%), 21 isolates harboring AAC (100%) and 20 isolates harboring GES (95.2%) -type enzymes (Table 4). MDR consistently correlated with the presence of
194 oxacillinases, mostly in the form of plasmid-mediated OXA-51 and OXA-23 enzymes which
195 were detected in 21 (100%) and 13 (61.9%) of the clinical isolates collected, respectively. None
196 of the isolates harbored OXA-58, OXA-24 or OXA-48. ISAba1 was detected in 16 (76.2%)
197 isolates. None of the clinical isolates harbored KPC, IMP, VIM, SIM, NDM, or QNR-type genes
198 (Table 4).
200 3.3. Frequency of Insertion Sequences for Different Enzymes
201 The frequency of insertion sequences presence in all of the clinical isolates of A. baumannii
202 harboring antibiotic resistance genes were found to be 7 out of 12 (58.3%) in OXA-23, 7 out of 7
203 (100%) in PER, 15 out of 20 (75%) in GES and 17 out of 21 (81%) in AAC (Table 4).
205 3.4. Molecular Genotyping of A. baumannii Clinical Isolates
206 Genotyping analysis of the 21 clinical isolates by PFGE revealed the circulation of different
207 PFGE types. We found high clonal relationship among all the typed strains. All the isolates were
208 MDR to at least three antimicrobial groups. PFGE analysis demonstrated that the majority of
209 clinical isolates are highly related (>85%). MLST studies have shown that 5 (23.8%) clinical
210 isolates of A. baumannii were found to be related to sequence type-1 (ST1) and 16 (76.2%)
211 belong to sequence type-2 (ST2) (Table 5). The results of PFGE and MLST are summarized in
212 Figure 1, along with the information of the specimen original sources.
214 3.5. Plasmid Curing of A. baumannii Clinical Isolates
215 Our results have shown that the five clinical isolates cured for plasmids, did not lose their ability
216 for resistance to antimicrobial agents. Plasmids curing using temperature as a curing agent failed
217 even after 3 days incubation at 47oC (Table S1).
4. Discussion:
Antimicrobial resistance in Enterobacteriaceae has emerged as a major clinical problem in recent years.42'43 Drug resistance among this group of bacteria is mainly caused by the emergence and proliferation of extended-spectrum P-lactamases,44 fluoroquinolone resistance,45 and the dissemination of multiple drug-resistant (MDR) and carbapenem resistant strains.46,47 Data from the National Healthcare Safety Network at the CDC showed high rates of carbapenem resistance among A. baumannii throughout USA with increased incidence in hospital-associated infections especially those of ventilated acquired pneumonia, central line associated bloodstream infections, catheter associated urinary tract infections and surgical site infections.48 Understanding the fundamental mechanisms that underline Acinetobacter infections including the original sources of the infecting strains, resistance patterns, their clonality and geographical spread are critical for the development of appropriate infection control measures and more efficient treatment strategies.
We used two typing methods of PFGE and MLST to detect the molecular epidemiology of A. baumannii isolates from Harbor-UCLA Medical Center in Los Angeles County.49,50 By using MLST we have shown that clinical isolates of A. baumannii belonged to two main clones; ST1 and ST2. The high clonal relationship in PFGE analysis between HUMC strains as reflected by > 85% is in agreement with the MLST studies which showed more than 76.2% of the tested isolates belonged to ST2. It is prudent to mention that MLST is a high resolution molecular tool for discriminating between closely related bacterial species.50 The first MLST scheme for A. baumannii was published by Bartual et al 5 and by Diancourt et al52 at the Pasteur Institute (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html), which we have used in
243 this study. Further, MLST approaches to genotype A. baumannii isolates, which are based on
244 sequencing regions of housekeeping genes,53 are reproducible and portable facilitating
245 comparison among laboratories worldwide. While MLST is an expensive typing method due to
246 the need for DNA sequencing, selective use of this technique can substantially enhance our
247 understanding of molecular epidemiology across different hospitals and geographic locations. In
248 contrast, although PFGE analysis is highly discriminatory, it is not suitable for inter-laboratory
249 comparisons unless the procedures are meticulously standardized,49 and the interpretation of the
250 pulsed field results may be a challenge in non-outbreak situations.50 However, genotyping by
251 methods like PFGE allows investigation of clonal spread and can be used to identify the source
252 of the original infection. Therefore, whenever possible the use of both methods for genotyping is
253 advisable. Equally important, our previous work using PFGE analysis on 5 isolates of MDR A.
254 baumannii demonstrated that all these isolates were genetically different from the drug
255 susceptible A. baumannii ATCC 17978.54
256 Previous genome sequencing studies have shown that MDR A. baumannii strains causing
257 infection are related to one another with extensive variation in gene content even among strains
258 that were very closely related phylogenetically and epidemiologically.55 Several mechanisms
259 contribute to this diversity, including transfer of mobile genetic elements and mobilization of
260 insertion sequences.55 In addition, widespread genetic variation among clinical isolates from the
261 same hospital and/or patient reinforces the need for molecular diagnostic testing and genomic
262 analysis to determine resistance profiles, rather than to rely primarily on strain typing and
263 antimicrobial resistance phenotypes for molecular epidemiological studies.55
The role of insertion sequences is important to understand the expression of carbapenemases in A. baumannii (e.g., ISAba/). It has been reported that insertion sequences play a role in the expression of the carbapenem-hydrolyzing P-lactamases40 In this study, we have shown that ISAba/ was detected in almost 76.2% of the clinical isolates. We also showed that not all isolates harboring ISAba/ were resistant to carbapenems (Table 4). These findings are concordant with the fact that resistance to carbapenems is mainly caused by the OXA-type enzymes, including plasmid-encoded P-lactamases (OXA-23, 0XA-40, and OXA-58).38 Studies have shown that chromosomally encoded OXA P-lactamase (OXA-51-like) can confer resistance to carbapenems in A. baumannii when the genetic environment around the gene promoted its expression.38,56 Our findings are in agreement with other studies in which we show that almost all A. baumannii strains possess chromosomally encoded OXA P-lactamase (OXA-51 like). The high frequency of insertion sequences for different types of enzymes, oxacillinases and carbapenemases series in A. baumannii could be due to differences in antibiotic treatment given to those patients. Our plasmid curing studies have shown that clinical strains did not lose their ability for resistance to antimicrobial agents, indicating a high stability of acquired plasmids.
The two clonal lineages of MDR A. baumannii identified in our study have been found in other States as well.46 In the United States, an outbreak of MDR A. baumannii in Houston was caused by clonal complex 92 (an ST-2 type).57 In addition, a survey of bacterial isolates collected from 52 U.S. hospitals over 6 years also showed the preponderance of the same clone.58 Other studies has revealed good correlation between antibiotic susceptibility profiles and genetic fingerprints from clinical A. baumannii isolates from nosocomial outbreaks and the mechanisms of antibiotic resistance.59 Molecular epidemiology of clinical isolates of A. baumannii identified in New York, Pennsylvania, Florida, Missouri, Nevada, and California revealed the
288 predominance of CC92 among carbapenem non-susceptible isolates in US hospitals, suggesting
289 that they constitute part of the global epidemic driven by this clonal complex belonging to
290 EUII.46 Worldwide, it is not surprising to detect common STs. For examples, ST-1 and ST-2
291 have been previously identified in the Middle East including Saudi Arabia, Lebanon and
292 Yemen.60-62 Moreover, ST-1 and ST-2 are known as endemic strains in European countries
293 including Spain, Italy, France and Greece,63-66 Asia including Japan and Taiwan,67'68 and even
294 the Scandinavian countries like in Denmark.69
295 There is no doubt that early pathogen identification, followed by the right antibiotic
296 treatment may reduce the prevalence of antibiotic resistance in A. baumannii. Therefore, future
297 studies will focus on characterizing the composite transposable elements in detail. Though, the
298 clinical isolates collected in this study may not represent the overall epidemiology of MDR A.
299 baumannii since it all originated from one hospital in Los Angeles County, we plan to further
300 research the epidemiological analyses of this organism beside other MDR and XDR organisms in
301 other hospitals for continuous surveillance of these strains in the United States.
303 5. Conclusion:
305 Two distinct clones of MDR or XDR A. baumannii were identified at Harbor UCLA Medical
306 Center in Los Angeles County. The epidemiological data obtained suggested that the increase in
307 the number of A. baumannii infections in that hospital was caused by these two clones. MLST
308 studies maybe more accurate in distinguishing between A. baumannii isolates than PFGE typing.
309 This study supports a more complete understanding of genotyping of antibiotic resistance for
better assessment of MDR and/or XDR strains transmission. Continuous surveillance is needed for monitoring the spread of these strains equipped with multiple drug resistance mechanisms.
Competing Interests:
The authors declare that there are no competing interests. Authors' Contributions:
SS, AD, and ASI conceived and designed the experiments. SS, LV and MB performed the experiments. SS, LV and ASI analyzed the data. SS and ASI wrote the paper. AD, LV and MB revised the paper. All authors have approved the final article.
Acknowledgments:
This work was supported by Public Health Service grants 1R21AI119339-01 to ASI. The authors would like to extend sincere appreciation to Dr. Brad Spellberg for providing the clinical information of the isolates at Harbor-UCLA Medical Center and Mr. Shady Farran at Research Core Facility of the Health Sciences Centre, Kuwait University (Project No. SRUL02/13), Mrs. Qudsiya Electricwala and Mrs. Leina Ibrahim at Faculty of Allied Health Sciences, Kuwait University for their help and assistance.
Research described in this manuscript was conducted in part at the research facilities of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center.
334 Table 1: Gene primers used for housekeeping genes detection by PCR in genes in clinical A.
335 baumannii isolates in MLST studies.
Gene Primer Sequence Amplicon Size
cpn60-F cpn60-R ACTGTACTTGCTCAAGC TTCAGCGATGATAAGAAGTGG 405 bp
fusA-F fusA-R ATCGGTATTTCTGCKCACATYGAT CCAACATACKYTGWACACCTTTGTT 633 bp
gltA-F gltA-R AATTTACAGTGGCACATTAGGTCCC GCAGAGATACCAGCAGAGATACACG 483 bp
pyrG-F pyrG-R GGTGTTGTTTCATCACTAGGWAAAGG ATAAATGGTAAAGAYTCGATRTCACCMA 297 bp
recA-F recA-R CCTGAATCTTCYGGTAAAAC GTTTCTGGGCTGCCAAACATTAC 372 bp
rplB-F rplB-R GTAGAGCGTATTGAATACGATCCTAACC CACCACCACCRTGYGGGTGATC 330 bp
rpoB-F rpoB-R GGTCCTGGTGGTTTAACACG CGAATAACGATACGAGAAGCA 456 bp
338 Table 2: Gene primers used for PCR amplification of antibiotic resistance genes in clinical A.
339 baumannii isolates.
Gene Amplicon Size Tm °C Primer Sequence
OXA-58 599bp 52 AAGTATTGGGGCTTGTGCTG (Forward) CCCCTCTGCGCTCTACATAC (Reverse)
OXA-51 353bp 52 TAATGCTTTGATCGGCCTTG (Forward) TGGATTGCACTTCATCTTGG (Reverse)
OXA-24 246bp 52 GGTTAGTTGGCCCCCTTAAA (Forward) AGTTGAGCGAAAAGGGGATT(Reverse)
OXA-23 501bp 52 GATCGGATTGGAGAACCAGA (Forward) ATTCTGACCGCATTTCCAT(Reverse)
OXA-48 438bp 62 GCGTGGTTAAGGATGAACAC (Forward) CATCAAGTTCAACCCAACCG (Reverse)
CTXM 550bp 60 CGCTTTGCGATGTGCAG (Forward) ACCGCGATATCGTTGGT (Reverse)
CTXM2 896bp 55 CGGAATTCATGATGACTCAGAGCATTCG (Forward) GCTCTAGATTATTGCATCAGAAACCGTG (Reverse)
PER 900bp 43 If ATGAATGTCATTATAAAAGC (Forward) AATTTGGGCTTAGGGCAGAA (Reverse)
VEB 600bp 55 CGACTTCCATTTCCCGATGC (Forward) GGACTCTGCAACAAATACGC (Reverse)
QnrA 580bp 54 AGAGGATTTCTCACGCCAGG (Forward) TGCCAGGCACAGATCTTGAC (Reverse)
QnrS 428bp 54 GCAAGTTCATTGAACAGGGT (Forward) TCTAAACCGTCGAGTTCGGCG (Reverse)
QnrB 264bp 54 GGMATHGAAATTCGCCACTG (Forward) TTTGCYGYYCGCCAGTCGAA (Reverse)
TEM 850bp 42 GAGTATTCAACATTTCCGTGTC (Forward) TAATCAGTGAGGCACCTATCTC (Reverse)
GES 846bp 55 ATGCGCTTCATTCACGCAC (Forward)
CTATTTGTCCGTGCTCAGGA (Reverse)
SHV 861bp 55 ATGCGTTATWTTCGCCTGTGT (Forward) TTAGCGTTGCCAGTGCTCG (Reverse)
CTX 554bp 60 TCTTCCAGAATAAGGAATCCC (Forward) CCGTTTCCGCTATTACAAAC (Reverse)
GyrA6 620bp 56 CGACCTTGCGAGAGAAAT (Forward) GTTCCATCAGCCCTTCAA (Reverse)
ParCF43 964bp 53 AGCGCCTTGCGTACATGAAT (Forward) GTGGTAGCGAAGAGGTGGTT (Reverse)
AAC 482bp 55 TTGCGATGCTCTATGAGTGGCTA (Forward) CTCGAATGCCTGGCGTGTTT (Reverse)
SIM 570bp 65 TACAAGGGATTCGGCATCG (Forward) TAATGGCCTGTTCCCATGTG (Reverse)
IMP 232bp 60 GGAATAGAGTGGCTTAAYTC (Forward) TCGGTTTAAYAAAACAACCACC (Reverse)
VIM 390bp 62 GATGGTGTTTGGTCGCATA (Forward) CGAATGCGCAGCACCAG (Reverse)
NDM 621bp 65 GGTTTGGCGATCTGGTTTTC (Forward) CGGAATGGCTCATCACGATC (Reverse)
KPC 798bp 58 CGTCTAGTTCTGCTGTCTTG (Forward) CTTGTCATCCTTGTTAGGCG (Reverse)
IS 615bp 56 GTGCCCAAGGGGAGTGTATG (Forward) ACYTTACTGGTRCTGCACAT (Reverse)
ISAbaI 389bp 57 ATGCAGCGCTTCTTTGCAGG (Forward) AATGATTGGTGACAATGAAG (Reverse)
342 Table 3: Susceptibility profiles of HUMC strains. Isolates were designated susceptible (S),
343 intermediate (I), or resistant (R) according to CLSI antibiotic breakpoint guidelines. Minimum
344 inhibitory concentrations (MIC) were shown in brackets.
Antibiotic/
Isolate A/S AK CAZ CP CPE GM IMP LVX MER T/S TE TIM TO CST TGC
I S R R R R R R R R I R R S S
HUMC-1 (16/8) (<16) (>32) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (<1)
R R R R R R R R R R I R R S R
HUMC-3 (>16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (4)
I S R R R R R R R R I R S S I
HUMC-4 (16/8) (<16) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (<4) (<2) (2)
R R R R R R S R I R R R R S S
HUMC-5 (>16/8) (>32) (>16) (>2) (>16) (>8) (<4) (>4) (8) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R R R S R I R R R R S S
HUMC-6 (>16/8) (>32) (>16) (>2) (>16) (>8) (<4) (>4) (8) (>2/38) à (>8) (>64) (>8) (<2) (<1)
S S R R R R I R R R I R R S I
HUMC-9 (<8/4) (<16) (>16) (>2) (>16) (>8) (8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (2)
S R R R R R S R S R R R R S S
HUMC-10 (<8/4) (>32) (>16) (>2) (>16) (>8) (<4) (>4) (<4) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R I R S R I R R R R S S
HUMC-11 (>16/8) (>32) (>16) (>2) (16) (>8) (<4) (>4) (8) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R R R S R S R R R R S S
HUMC-12 (>16/8) (>32) (>16) (>2) (>16) (>8) (<4) (>4) (<4) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R R R S R I R R R R S S
HUMC-13 (>16/8) (>32) (>16) (>2) (>16) (>8) (<4) t (>4) (8) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R R R S R I R R R R S S
HUMC-14 (>16/8) (>32) (>16) (>2) (>16) (>8) (<4) (>4) (8) (>2/38) (>8) (>64) (>8) (<2) (<1)
I R R R R R R R R R I R R S R
HUMC-15 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (4)
I R R R R R R R R S I R R S S
HUMC-16 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (<2/38) (8) (>64) (>8) (<2) (<1)
I R R R R R R R R R R R R S S
HUMC-17 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (>8) (>64) (>8) (<2) (<1)
I R R R R R R R R R I R R S I
HUMC-18 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (2)
S R R R R R R R R R R R R S S
HUMC-19 (<8/4) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (>8) (>64) (>8) (<2) (<1)
I R R R R R R R R R R R R S S
HUMC-20 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (>8) (>64) (>8) (<2) (<1)
I R R R R R R R R R R R R R S
HUMC-21 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (>8) (>64) (>8) (>2) (<1)
I R R R R R R R R R I R R S S
HUMC-22 (16/8) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (8) (>64) (>8) (<2) (<1)
S R R R R R R R R R R R R S S
HUMC-23 (<8/4) (>32) (>16) (>2) (>16) (>8) (>8) (>4) (>8) (>2/38) (>8) (>64) (>8) (<2) (<1)
R R R R I R S R I R R R R S S
HUMC-24 (>16/8) (>32) (>16) (>2) (16) (>8) (<4) (>4) (8) (>2/38) (>8) (>64) (>8) (<2) (<1)
Abbreviations: A/S, Ampicillin/Sulbactam; AK, Amikacin; CAZ, Ceftazidime; CP, Ciprofloxacin; CPE, Cefepime; GM, Gentamicin; IMP, Imipenem; LVX, Levofloxacin; MER, Meropenem; T/S, Trimethoprim/Sulfamethoxazole; TE, Tetracycline; TIM, Ticarcillin/K Clavulanate; TO, Tobramycin; CST, Colistin; TGC, Tigecycline.
349 Table 4: Antibiotic resistance genes results detected by PCR in HUMC clinical strains.
350 Abbreviations: (-) denotes negative, (+) denotes positive PCR reaction.
Gene/ Isolate OXA-51 OXA-23 PER GES AAC ISAba1
HUMC-1 + + - + + +
HUMC-3 + + - + + -
HUMC-4 + - + + + +
HUMC-5 + - + + + +
HUMC-6 + - + + + +
HUMC-9 + + - + + +
HUMC-10 + - - + + +
HUMC-11 + - + + + +
HUMC-12 + - - + + +
HUMC-13 + - + + + +
HUMC-14 + - + + + +
HUMC-15 + + - + + -
HUMC-16 + + - + + +
HUMC-17 + + - + + +
HUMC-18 + + - + + -
HUMC-19 + + - - + +
HUMC-20 + + - + + +
HUMC-21 + + - + + -
HUMC-22 + + - + + -
HUMC-23 + + - + + +
HUMC-24 + - + + + +
360 Table 5. Allele and sequence number results of HUMC clinical isolates as analyzed by MLST.
HUMC Strains Allele Number ST
Cpn60 fusA gltA pyrG recA rplB rpoB
HUMC-1 2 2 2 2 2 2 2 2
HUMC-3 1 1 1 1 5 1 1 * 1
HUMC-4 2 2 2 2 2 2 2 2
HUMC-5 2 2 2 2 2 2 2 2
HUMC-6 2 2 2 2 2 2 2 2
HUMC-9 2 2 2 2 2 2 2 2
HUMC-10 2 2 2 2 2 2 2 2
HUMC-11 2 2 2 2 2 2 2 2
HUMC-12 2 2 2 2 2 2 2 2
HUMC-13 2 2 2 2 2 2 2 2
HUMC-14 2 2 2 2 2 2 2 2
HUMC-15 1 1 1 1 5 1 1 1
HUMC-16 1 1 1 1 5 1 1 1
HUMC-17 2 2 2 2 2 2 2 2
HUMC-18 1 1 1 1 5 1 1 1
HUMC-19 2 2 2 2 2 2 2 2
HUMC-20 2 2 2 2 2 2 2 2
HUMC-21 2 2 2 2 2 2 2 2
HUMC-22 1 1 1 1 5 1 1 1
HUMC-23 2 2 2 2 2 2 2 2
HUMC-24 2 2 2 2 2 2 2 2
[□51 qij iV fii IP
Figure 1: Dendrogram representing PFGE profiles, MLST results and site of infection of HUMC clinical isolates.
PFGE-ApaI
65 70 7 80 85 90 95
PFGE-ApaI
HUMC-12
HUMC-10
HUMC-6
HUMC-5
HUMC-14
HUMC-13
HUMC-24
HUMC-11
HUMC-19
HUMC-23
HUMC-1
HUMC-9
HUMC-21
HUMC-20
HUMC-22
HUMC-17
HUMC-16
HUMC-4
HUMC-3
HUMC-15
HUMC-18
2 2 2 2 2 2 2 2 2 2
2 2 1 1 2 1
Stump Foot Sputum BAL
Respiratory
Respiratory
Abdomen
Sputum
Respiratory
Respiratory
Sputum
Abdomen
Respiratory
Respiratory Respiratory
Sputum Wound Respiratory Respiratory
382 References:
383 1. Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of
384 multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2007;
385 51:3471-84.
387 2. Maragakis LL, Tucker MG, Miller RG, Carroll KC, Perl TM. Incidence and prevalence
388 of multidrug-resistant Acinetobacter using targeted active surveillance cultures. JAMA
389 2008; 299:2513-4.
391 3. Maragakis LL, Winkler A, Tucker MG, et al. Outbreak of multidrug-resistant Serratia
392 marcescens infection in a neonatal intensive care unit. Infect Control Hosp Epidemiol
393 2008; 29:418-23.
395 4. Paterson DL. The epidemiological profile of infections with multidrug-resistant
396 Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis 2006; 43 Suppl
397 2:S43-8.
399 5. Aronson NE, Sanders JW, Moran KA. In harm's way: infections in deployed American
400 military forces. Clin Infect Dis 2006; 43:1045-51.
402 6. Harman DR, Hooper TI, Gackstetter GD. Aeromedical evacuations from Operation Iraqi
403 Freedom: a descriptive study. Mil Med 2005; 170:521-7.
405 7. Hinsley DE, Phillips SL, Clasper JS. Ballistic fractures during the 2003 Gulf conflict
406 early prognosis and high complication rate. JR Army Med Corps 2006; 152:96-101.
408 8. Murray CK, Roop SA, Hospenthal DR, et al. Bacteriology of war wounds at the time of
409 injury. Mil Med 2006; 171:826-9.
Murray CK, Yun HC, Griffith ME, et al. Recovery of multidrug-resistant bacteria from combat personnel evacuated from Iraq and Afghanistan at a single military treatment facility. Mil Med 2009; 174:598-604.
Perez F, Hujer AM, Hulten EA, et al. Antibiotic resistance determinants in Acinetobacter spp and clinical outcomes in patients from a major military treatment facility. Am J Infect Control 2010; 38:63-5.
Talbot GH, Bradley J, Edwards JE, Jr., Gilbert D, Scheld M, Bartlett JG. Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clin Infect Dis 2006; 42:657-68.
Boucher HW, Talbot GH, Bradley JS, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009; 48:1-12.
Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008; 21(3):538-82. Review.
Dizbay M, Tunccan OG, Sezer BE, Hizel K. Nosocomial imipenem-resistant Acinetobacter baumannii infections: epidemiology and risk factors. Scand J Infect Dis 2010; 42:741-6.
Sunenshine RH, Wright MO, Maragakis LL, et al. Multidrug-resistant Acinetobacter infection mortality rate and length of hospitalization. Emerg Infect Dis 2007; 13:97-103.
Doi Y, Husain S, Potoski BA, McCurry KR, Paterson DL. Extensively drug-resistant Acinetobacter baumannii. Emerg Infect Dis 2009; 15:980-2.
Lautenbach E, Synnestvedt M, Weiner MG, et al. Epidemiology and impact of imipenem resistance in Acinetobacter baumannii. Infect ControlHosp Epidemiol 2009; 30:1186-92.
442 18. Metan G, Sariguzel F, Sumerkan B. Factors influencing survival in patients with multi-
443 drug-resistant Acinetobacter bacteraemia. Eur J Intern Med 2009; 20:540-4.
445 19. Gordon NC, Wareham DW. A review of clinical and microbiological outcomes following
446 treatment of infections involving multidrug-resistant Acinetobacter baumannii with
447 tigecycline. JAntimicrob Chemother 2009; 63:775-80.
449 20. Karageorgopoulos DE, Falagas ME. Current control and treatment of multidrug-resistant
450 Acinetobacter baumannii infections. Lancet Infect Dis 2008; 8:751-62.
452 21. Gardiner D, Dukart G, Cooper A, Babinchak T. Safety and efficacy of intravenous
453 tigecycline in subjects with secondary bacteremia: pooled results from 8 phase III clinical
454 trials. Clin Infect Dis 2010; 50:229-38.
456 22. Park YK, Jung SI, Park KH, et al. Independent emergence of colistin-resistant
457 Acinetobacter spp. isolates from Korea. Diagn Microbiol Infect Dis 2009; 64:43-51.
459 23. Adams MD, Nickel GC, Bajaksouzian S, et al. Resistance to colistin in Acinetobacter
460 baumannii associated with mutations in the PmrAB two-component system. Gardiner D,
461 Dukart G, Cooper A, Babinchak T. Safety and efficacy of intravenous tigecycline in
462 subjects with secondary bacteremia: pooled results from 8 phase III clinical trials. Clin
463 Infect Dis 2009; 53:3628-34.
465 24. Falagas ME, Rafailidis PI, Matthaiou DK, Virtzili S, Nikita D, Michalopoulos A.
466 Pandrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter
467 baumannii infections: characteristics and outcome in a series of 28 patients. Int J
468 Antimicrob Agents 2008; 32:450-4.
470 25. Levin AS, Barone AA, Penco J, et al. Intravenous colistin as therapy for nosocomial
471 infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter
472 baumannii. Clin Infect Dis 1999; 28:1008-11.
Oliveira MS, Prado GV, Costa SF, Grinbaum RS, Levin AS. Ampicillin/sulbactam compared with polymyxins for the treatment of infections caused by carbapenem-resistant Acinetobacter spp. JAntimicrob Chemother 2008; 61:1369-75.
Valencia R, Arroyo LA, Conde M, et al. Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter baumannii in a tertiary care university hospital. Infect ControlHosp Epidemiol 2009; 30:257-63.
Pogue JM, Mann T, Barber KE, Kaye KS. Carbapenem-resistant Acinetobacter baumannii: epidemiology, surveillance and management. Expert Rev Anti Infect Ther 2013 Apr; 11(4):383-93. Review.
Clinical and Laboratory Standards Institute, 2012. Performance standards for antimicrobial susceptibility testing; twenty-first informational supplement. Document M100-S21. CLSI, Wayne, PA 501 2012
Blanco M, Alonso MP, Nicolas-Chanoine MH, Dahbi G, Mora A, Blanco JE, López C, Cortés P, Llagostera M, Leflon-Guibout V, Puentes B, Mamani R, Herrera A, Coira MA, García-Garrote F, Pita JM, Blanco J. Molecular epidemiology of Escherichia coli producing extended-spectrum beta-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15. J Antimicrob Chemother 2009; 63(6):1135-41. Epub 2009 Apr 7.
Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P. Emergence of metallo-P-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia. Antimicrob Agents Chemother 2010; 54(11):4914-6. Epub 2010 Sep 7.
Cattoir V, Poirel L, Rotimi V, Soussy CJ, Nordmann P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother 2007; 60(2):394-7. Epub 2007 Jun 8.
505 33. Coelho A, González-López JJ, Miró E, Alonso-Tarrés C, Mirelis B, Larrosa MN,
506 Bartolomé RM, Andreu A, Navarro F, Johnson JR, Prats G. Characterization of the CTX-
507 M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan
508 area: plasmid diversity and chromosomal integration. Int J Antimicrob Agents 2010;
509 36(1):73-8. Epub 2010 Apr 13.
511 34. Giraud E, Brisabois A, Martel JL, Chaslus-Dancla E. Comparative studies of mutations in
512 animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp.
513 suggest a counter selection of highly fluoroquinolone-resistant strains in the field.
514 Antimicrob Agents Chemother 1999; 43(9):2131-7.
516 35. Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes
517 in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40(6):2153-62.
519 36. Poirel L, Bonnin RA, Nordmann P. Genetic support and diversity of acquired extended-
520 spectrum P-lactamases in Gram-negative rods. Infect Genet Evol 2012; 12(5):883-93.
521 Epub 2012 Mar 3.
523 37. Poirel L, Naas T, Nordmann P. Diversity, epidemiology, and genetics of class D P-
524 lactamases. Antimicrob Agents Chemother 2010; 54(1):24-38. Epub 2009 Aug 31.
525 Review.
527 38. Evans BA, Amyes SG. OXA P-lactamases. Clin Microbiol Rev 2014; 27(2):241-63.
529 39. Kiratisin P, Apisarnthanarak A, Saifon P, Laesripa C, Kitphati R, Mundy LM. The
530 emergence of a novel ceftazidime-resistant CTX-M extended-spectrum beta-lactamase,
531 CTX-M-55, in both community-onset and hospital-acquired infections in Thailand.
532 Diagn Microbiol Infect Dis 2007; 58(3):349-55. Epub 2007 Apr 20.
Mugnier PD, Poirel L, Nordmann P. Functional analysis of insertion sequence ISAbal, responsible for genomic plasticity of Acinetobacter baumannii. J Bacteriol 2009; 191(7):2414-8.
Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne PathogDis 2006; 3(1):59-67.
Bao L, Peng R, Ren X, Ma R, Li J, Wang Y. Analysis of some common pathogens and their drug resistance to antibiotics. Pak J Med Sci 2013; 29(1):135-9.
Dashti AA, Vali L, El-Shazly S, Jadaon MM. The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait. BMC Microbiol 2014; 28; 14:214. doi: 10.1186/s12866-014-0214-6.
Sharma M, Pathak S, Srivastava P. Prevalence and antibiogram of Extended Spectrum ß-Lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. J Clin Diagn Res 2013;7(10):2173-7. Epub 2013 Oct 5.
Sjölund-Karlsson M, Howie R, Rickert R, Krueger A, Tran TT, Zhao S, Ball T, Haro J, Pecic G, Joyce K, Fedorka-Cray PJ, Whichard JM, McDermott PF. Plasmid-mediated quinolone resistance among non-Typhi Salmonella enterica isolates, USA. Emerg Infect Dis 2010; 16(11):1789-91.
Adams-Haduch JM, Onuoha EO, Bogdanovich T, Tian GB, Marschall J, Urban CM, Spellberg BJ, Rhee D, Halstead DC, Pasculle AW, Doi Y. Molecular epidemiology of carbapenem-nonsusceptible Acinetobacter baumannii in the United States. J Clin Microbiol 2011; 49(11):3849-54.
Lee GC, Lawson KA, Burgess DS. Clinical epidemiology of carbapenem-resistant enterobacteriaceae in community hospitals: a case-case-control study. Ann Pharmacother 2013; 47(9):1115-21.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK; National Healthcare Safety Network Team; Participating National Healthcare Safety Network Facilities. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 20062007. Infect Control Hosp Epidemiol 2008 Nov; 29(11):996-1011. doi: 10.1086/591861. Erratum in: Infect Control Hosp Epidemiol 2009; 30(1):107.
Seifert H, Dolzani L, Bressan R, van der Reijden T, van Strijen B, Stefanik D, Heersma H, Dijkshoorn L. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. J Clin Microbiol 2005; 43(9):4328-35.
Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ. Application of molecular techniques to the study of hospital infection. Clin Microbiol Rev 2006; 19(3):512-30.
Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 2005; 43(9):4382-90. Erratum in: J Clin Microbiol 2007 Jun; 45(6):2101.
Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One 2010; 7; 5(4):e10034.
Urwin R and Maiden MC. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 2003; 11(10):479-87.
54. Luo G, Spellberg B, Gebremariam T, Lee H, Fu Y, French SW, Ibrahim AS. Diabetic murine models for Acinetobacter baumannii infection. J Antimicrob Chemother. 2012; doi:10.1093/jac/dks050.
55. Wright MS, Haft DH, Harkins DM, Perez F, Hujer KM, Bajaksouzian S, Benard MF, Jacobs MR, Bonomo RA, Adams MD. New insights into dissemination and variation of the health care-associated pathogen Acinetobacter baumannii from genomic analysis. M Bio 2014; 21; 5(1):e00963-13.
56. Opazo A, Vali L, Al Obaid K, Dashti AA, Amyes SG. Novel genetic structure harbouring blaPER-1 in ceftazidime-resistant Acinetobacter baumannii isolated from Kuwait. Int J Antimicrob Agents 2014; 43(4):383-4. doi: 10.1016/j.ijantimicag.2014.01.001. Epub 2014 Jan 21.
57. Shelburne SA 3rd, Singh KV, White AC Jr, Byrne L, Carmer A, Austin C, Graviss E, Stager C, Murray BE, Atmar RL. Sequential outbreaks of infections by distinct Acinetobacter baumannii strains in a public teaching hospital in Houston, Texas. J Clin Microbiol. 2008 Jan; 46(1):198-205. Epub 2007 Nov 14.
58. Wisplinghoff H, et al. MLST-based molecular epidemiology of clinical Acinetobacter baumannii bloodstream isolates from 52 hospitals in the US, 2010. Abstr. C2-590. Abstr. 50th Intersci Conf Antimicrob. Agents Chemother.
59. Valentine SC, Contreras D, Tan S, Real LJ, Chu S, Xu HH. Phenotypic and molecular characterization of Acinetobacter baumannii clinical isolates from nosocomial outbreaks in Los Angeles County, California. J Clin Microbiol. 2008 Aug; 46(8):2499-507. doi: 10.1128/JCM.00367-08. Epub 2008 Jun 4.
60. Aly M, Tayeb HT, AlJohani SM, Alyamani EJ, Aldughaishem F, Alabdulkarim I, et al. Genetic diversity of OXA-51-like genes among multidrug-resistant Acinetobacter
627 baumannii in Riyadh, Saudi Arabia. Eur J Clin Microbiol Infect Dis. 2014 33, 1223628 1228.doi:10.1007/s10096-014-2068-0
630 61. Rafei R, Dabboussi F, Hamze M, Eveillard M, Lemarie C, Gaultier MP, Mallat H,
631 Moghnieh R, Husni-Samaha R, Joly-Guillou ML, Kempf M. Molecular analysis of
632 Acinetobacter baumannii strains isolated in Lebanon using four different typing methods.
633 PLoS One. 2014 Dec 26; 9(12):e115969. doi: 10.1371/journal.pone.0115969. eCollection
634 2014.
636 62. Bakour S, Alsharapy SA, Touati A, and Rolain, JM. Characterization of Acinetobacter
637 baumannii clinical isolates carrying bla (OXA-23) carbapenemase and 16S rRNA
638 methylase armA genes in Yemen. Microb Drug Resist. 2014 20, 604639 609.doi:10.1089/mdr.2014.0018
641 63. Villalon P, Valdezate S, Medina-Pascual MJ, Rubio V, Vindel A and Saez Nieto JA.
642 Clonal diversity of nosocomial epidemic Acinetobacter baumannii strains isolated in
643 Spain. J Clin Microbiol, 2011. 49, 875-882.doi: 10.1128/JCM.01026-10
645 64. Mezzatesta ML, Caio C, Gona F, Cormaci R, Salerno I, Zingali T, et al. Carbapenem and
646 multidrug resistance in Gram-negative bacteria in a single centre in Italy: considerations
647 on in vitro assay of active drugs. Int J Antimicrob Agents, 2014. 44, 112648 116.doi:10.1016/j.ijantimicag.2014. 04.014
650 65. Jeannot K, Diancourt L, Vaux S, Thouverez M, Ribeiro A, Coignard B, Courvalin P,
651 Brisse S. Molecular epidemiology of carbapenem non-susceptible Acinetobacter
652 baumannii in France. PLoS One. 2014 Dec 17; 9(12):e115452. doi:
653 10.1371/journal.pone.0115452. eCollection 2014.
655 66. Gogou V, Pournaras S, Giannouli M, Voulgari E, Piperaki ET, Zarrilli R, Tsakris A.
656 Evolution of multidrug-resistant Acinetobacter baumannii clonal lineages: a 10 year
657 study in Greece (2000-09). J Antimicrob Chemother. 2011 Dec; 66(12):2767-72. doi:
658 10.1093/jac/dkr390. Epub 2011 Sep 19.
660 67. Matsui M, Suzuki S, Yamane K, Suzuki M, Konda T, Arakawa Y, Shibayama K.
661 Distribution of carbapenem resistance determinants among epidemic and non-epidemic
662 types of Acinetobacter species in Japan. J Med Microbiol. 2014 Jun; 63(Pt 6):870-7. doi:
663 10.1099/jmm.0.069138-0. Epub 2014 Mar 5.
665 68. Chen CM, Ke SC, Li CR, Chang CC. The comparison of genotyping, antibiogram, and
666 antimicrobial resistance genes between carbapenem-susceptible and -resistant
667 Acinetobacter baumannii. Comp Immunol Microbiol Infect Dis. 2014 Dec; 37(5-6):339-
668 46. doi: 10.1016/j.cimid. 2014.10.002. Epub 2014 Oct 17.
670 69. Hammerum AM, Hansen F, Skov MN, Stegger M, Andersen PS, Holm A, Jakobsen L,
671 Justesen US. Investigation of a possible outbreak of carbapenem-resistant Acinetobacter
672 baumannii in Odense, Denmark using PFGE, MLST and whole-genome-based SNPs. J
673 Antimicrob Chemother. 2015 Jul; 70(7):1965-8. doi: 10.1093/jac/dkv072. Epub 2015 Mar
674 19.
