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. 2021 Apr 29;16(4):e0250896. doi: 10.1371/journal.pone.0250896

Multidrug resistance pattern of Acinetobacter species isolated from clinical specimens referred to the Ethiopian Public Health Institute: 2014 to 2018 trend anaylsis

Zeleke Ayenew 1,*, Eyasu Tigabu 1,2, Elias Syoum 1, Semira Ebrahim 1, Dawit Assefa 1, Estifanos Tsige 1
Editor: Monica Cartelle Gestal3
PMCID: PMC8084144  PMID: 33914829

Abstract

Background

Acinetobacter species have been a leading cause of nosocomial infections, causing significant morbidity and mortality over the entire world including Ethiopia. The most important features of A. baumannii are its ability to persist in the hospital environment and rapidly develop resistance to a wide variety of antibiotics. This study aimed to determine trend of antimicrobial resistance in Acinetobacter species over a five years period.

Method

A retrospective data regarding occurrence and antimicrobial resistance of Acinetobacter species recovered from clinical specimens referred to the national reference laboratory was extracted from microbiology laboratory data source covering a time range from 2014 to 2018. Socio-demographic characteristics and laboratory record data was analyzed using SPSS 20.

Results

A total of 102 strains of Acinetobacter species were analyzed from various clinical specimens. Majority of them were from pus (33.3%) followed by blood (23.5%), urine (15.6%) and body fluid (11.7%). Significant ascending trends of antimicrobial resistance was shown for meropenem (12.5% to 60.7%), ceftazidime (82.1% to 100%), ciprofloxacin (59.4% to 74.4%), ceftriaxone (87.1% to 98.6%), cefepime (80.0% to 93.3%) and pipracillin- tazobactam (67.8% to 96.3%). However, there was descending trend of antimicrobial resistance for tobramycin (56.5% to 42.8%), amikacin (42.1% to 31.4%) and trimethoprim-sulfamethoxazole (79.0 to 68.2%). The overall rate of carbapenem non-susceptible and multidrug resistance rates in Acinetobacter species were 56.7% and 71.6%.respectively.

Conclusion

A five year antimicrobial resistance trend analysis of Acinetobacter species showed increasing MDR and resistance to high potent antimicrobial agents posing therapeutic challenge in our Hospitals and health care settings. Continuous surveillance and appropriate infection prevention and control strategies need to be strengthened to circumvent the spread of multidrug resistant pathogens in health care facilities.

Introduction

Acinetobacter species are aerobic gram-negative bacilli that can cause healthcare-associated infections and can survive for prolonged periods in the environment and on the hands of healthcare workers [1]. Acinetobacter was first described in 1911 as Micrococcus calcoaceticus by Beijerinck, a Dutch microbiologist who isolated the organism from soil. Since then, it has had several names, nowadays known as Acinetobacter since the 1950s [2,3]. The genus Acinetobacter consists of more than 30 species, of which A. baumannii, and to a lesser extent genomic species 3 and 13TU, are mostly associated with clinical environment and nosocomial infections [3].

According to most recent scientific literature, Acinetobacter species are the second most common non-fermenting gram negative pathogens isolated from clinical specimens after Pseudomonas aeruginosa [2]. Acinetobacter baumannii has become increasingly responsible for causing hospital acquired infections (HAI), particularly in intensive care units (ICUs) [4]. It has been isolated from blood, sputum, skin, pleural fluid, and urine, usually in device associated infections [5]. The species are excellent biofilm producing bacteria, which facilitate their survival in hospital environments and are frequently found on the skin and in the respiratory and urinary tracts of hospitalized patients [6].

Acinetobacter baumannii is one of the most challenging pathogens among ESKAPE pathogens, standing for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A.baumannii, P.aeruginosa, and Enterobacteriaceae, capable of “escaping” from common antibacterial treatments due to its particular antibiotic resistance [7]. The bacteria produce naturally occurring AmpC β-lactamases, as well as naturally occurring oxacillinases (OXAs) with carbapenemase activity [8].

Multi-drug resistance (MDR) in Acinetobacter species is defined as non-susceptible to at least 1 agent in ≥3 antimicrobial categories [9]. The species are becoming increasingly resistant to nearly all routinely prescribed antimicrobial agents, including aminoglycosides, fluoroquinolones, and broad-spectrum β-lactams. The majority of strains are resistant to cephalosporin class of antimicrobials and resistance to carbapenems is increasingly reported [10]. Carbapenems which were once the mainstay therapy are no longer effective in controlling the infections caused by this organism. The foremost implication of infection with carbapenem resistant A. baumannii is the need to use "last-line" antibiotics such as colistin, polymyxin B, or tigecycline [11]. Sulbactam, a β-lactamase inhibitor, has good in vitro activity against Acinetobacter species and has been used successfully for treating carbapenem-resistant strains [12].

Risk factors for multi-drug resistant Acinetobacter colonization and infection include prolonged length of hospital stay, exposure to central venous catheterization, urinary catheterization, prior exposure to strong antimicrobials, greater severity of illness, surgery and receipt of invasive procedures [13].

People who have weakened immune systems, chronic lung disease, or diabetes, hospitalized patients, especially very ill patients on a ventilator, those with a prolonged hospital stay, those who have open wounds, or any person with invasive devices like urinary catheters are at greater risk for Acinetobacter infection. The infection can spread to susceptible persons by person-to-person contact or contact with contaminated surfaces [14].

Globally, MDR strains of Acinetobacter species are causing lethal hospital outbreaks often characterized by high morbidity and mortality. Due to the emergence of colistin resistance MDR-A. baumannii in clinical setting, WHO labeled the organism as critical pathogen [15]. A Center for Disease Control and Prevention (CDC) report, in the United States, in 2013, highlighted MDR Acinetobacter as a serious threat that causes ≈7,000 infections and ≈500 deaths each year. Nearly half of the strains isolated from persons with healthcare-associated infections reported to the CDC National Healthcare Safety Network in 2014 were carbapenem-nonsusceptible. Infections with carbapenem-resistant A. baumannii have been associated with death rates as high as 52% [16]. In Ethiopia, even though many studies had been conducted on antimicrobial resistance, there is still limited data showing MDR Acinetobacter infections associated morbidity and mortality at national level. Therefore, this study would provide information on the pattern of multidrug resistant Acinetobacter species isolated from different clinical specimens.

Materials and methods

Study design

A retrospective study design was followed to determine the prevalence of multidrug resistance Acinetobacter species and a trend analysis of antimicrobial susceptibility pattern among clinical specimens referred to the national reference laboratory of the Ethiopian Public Health Institute.

Study period and area

The laboratory recorded data from 2014 to 2018 was analyzed from January 2019 to June 2019 in Addis Ababa. National clinical bacteriology and mycology reference laboratory under Ethiopian Public Health Institute is designed to receive microbiological specimens from St. Paulus Hospital, Aabet Hospital, Ras Desta Damtew Hospital, St. Peter Hospital, Yekatit Hospital, Minilik II Hospital, Federal Police Hospital, Alert Hospital and other health facilities of Addis Ababa city administration.

Inclusion criteria

The study included all Acinetobacter species isolated from all ages and microbiological specimens referred to national reference laboratory during the study period. However, incomplete clinical information on patients and antimicrobial susceptibility testing report that did not comply Clinical and Laboratory Standards Institute guideline was excluded.

Data extraction method

A laboratory recorded routine data in which all samples types analyzed was incorporated in the analysis. A standardized questionnaire was used to collect socio-demographic characteristics, clinical history, hospitalization and antibiotic treatment. All records of Acinetobacter species and antimicrobial susceptibility test was collected in a questionnaire and entered to SPSS version 20.

Statistical analysis

Statistical analysis was performed using the SPSS version 20. Chi-square test and descriptive statistics (cross tab, frequency and proportion) were used to compare the trend of antimicrobial resistance rate, prevalence of MDR Acinetobacter species and resistance rates in empirically treated and untreated patients. P-value less than 0.05 were considered statistically significant.

Ethical consideration

The study was conducted after ethical clearance was obtained from Ethiopian Public Health Institute (EPHI) scientific and ethical review committee (SERC). The IRB of EPHI has given us a waiver of consent to conduct the study.

Data quality assurance

Retrospective check on quality record of media preparation per manufacturer instruction and laboratory Standard Operating Procedures (SOP) followed was conducted. We verified whether media, regents and antimicrobial agents have met expiration date and quality control parameters per clinical laboratory standard institute (CLSI). In addition, performance of quality control strains ATCC and sample storage system in laboratory was assessed from past records.

Results

Specimen source and demographical characteristics

A total of 102 Acinetobacter strains were isolated from various clinical specimens. Sixty percent of them were from males and 40% of them were from females with a mean age of 30.79 (SD±19.18). Pus/wound was the major source of the isolates (33.3%), followed by blood (23.5%), urine (15.6%), body fluid (11.7%), ear (4.9%), cerebrospinal fluid (3.9%), tracheal aspirate (1.9%), sputum (0.9%) and throat (0.9%).

Regarding the specimen and isolate sources, the majority were from St. Paulus Hospital (30.4%) followed by Aabet Hospital (23.5%), Ras Desta Damtew Hospital (16.7%), St. Peter Hospital (9.8%), Yekatit 12 Hospital (4.9%), Minilik II Hospital (3.9%), Federal Police Hospital (2.9%), Alert Hospital (0.09%) and others from health facilities (6.8%). Acinetobacter species were mostly recovered from hospital acquired infection (HAI) (26.5%) followed by sepsis (20.5%) and surgical site infection (14.7%). The distribution of Acinetobacter species according to different clinical diagnosis of patients is indicated in Fig 1.

Fig 1. Distribution of Acinetobacter species in relation to clinical conditions.

Fig 1

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Empirical treatment practice

Among the total of 102 patients, 26.5% of them provided specimen without initiation of antimicrobial therapy while 73.5% of them have taken antibiotic treatment empirically. Ceftriaxone was the most frequently prescribed (15.7%) antibiotics followed by ciprofloxacin and meropenem (7.8%). In addition, 84.3% of the patients have taken combined drugs (two or more antibiotics) before providing biological specimen. Vancomycin and ceftazidime were the most combined drugs prescribed that accounted for 9.8% (10/102) as shown in Table 1.

Table 1. Empirical treatment status of patients practice in health care settings from 2014–2018, Ethiopia.

Trends of antibiotics prescription (n = 102)
Prescription of antibiotics Frequency Percent
No antibiotics 27 26.5
Gentamycin 4 3.9
gentamycin+ceftriaxone+Vancomycin 1 1.0
gentamycin+cotrimoxzole+Augmentinn 1 1.0
Cefepime 2 2.0
Meropenem 8 7.8
Meropenem +Vancomycin 3 2.9
Ceftriaxone 16 15.7
cefepime +ceftriaxone 1 1.0
ceftriaxone+ciprofloxacin+Vancomycin 4 3.9
ceftraixone+ciprofloxacin +cotrimoxazole+cefepime 1 1.0
ceftriaxone +Vancomycin 3 2.9
ceftraiaxone+Vancomycin +ceftazidime 1 1.0
Ciprofloxacin 8 7.8
Vancomycin 5 4.9
Cftazidime +Vancomycin 10 9.8
Augmentin 2 2.0
OTHERS 5 4.9
Total 102 100.0
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Trends of Antimicrobial resistance (AMR)

All the isolates were tested for antimicrobials recommended for non sacrolytic bacteria according to CLSI guidelines. The antimicrobial resistance among isolates from empirically treated patients was proportionally high compared to empirically untreated patients [Fig 2].

Fig 2. Antimicrobial resistance among strains isolated from empirically treated and untreated patients referred from health facilities to Ethiopian public health institute, 2014–2018.

Fig 2

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The trends of antimicrobial resistance in the last two years (2017 to 2018) were as follows.

Group A antimicrobial agents

An increasing trend of antimicrobial resistance among antibiotics appropriate for inclusion in a routine, primary testing panel as well as routine reporting of the result for Acinetobacter species was observed as follows: meropenem (12.5% to 60.7%), doripenem (50.0% to 55.5%), ceftazidime (82.1% to 100%), ciprofloxacin (59.4% to 74.4%), gentamycin (55.8% to 58.0%); however, a decreasing trend of resistance was observed for tobramycin (56.5% to 42.8%) [Fig 3]. In addition, the prevalence of carbapenem non susceptible Acinetobacter species in all tested specimen types was 56.7% (21/37).

Fig 3. Trends of antimicrobial resistance for first line drugs among the Acinetobacter species from 2014 to 2018.

Fig 3

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Group B antimicrobial agents

Antimicrobial resistance for second option of antibiotics have also shown ascending trend: ceftriaxone (87.1% to 88.6%), cefepime (80.0% to 93.3%), and pipracillin tazobactam (67.8% to 96.3%), Amikacin (42.1% to 31.4%), trimethoprim-sulfamethoxazole (79.0 to 68.2%) [Fig 4].

Fig 4. Trends of antimicrobial resistance for second option of drugs among the Acinetobacter species from 2014 to 2018.

Fig 4

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In vitro activity of meropenem and doirpenem against Acinetobacter species were effective from 2014–2016; however, the resistance rate increased from 2017 to 2018.

The overall trends of antimicrobial classes over period of time are shown in Table 2.

Table 2. Trend of antimicrobial resistance in Acinetobacter species from 2014 to 2018.
Antimicrobial classes Antimicrobials Disk content % Resistance per year
2014 2015 2016 2017 2018
Carbapenem Meropenum 10ug 0 0 0 12.5 60.7
Doripenum 10ug 0 0 0 50.0 55.5
Cephalosporin Ceftriaxone 30ug 0 57.1 100 87.1 98.6
Cefepime 30ug 0 0 100 80 93.3
Ceftazidime 30ug 100 75 100 82.1 100
Beta lactams Piperacillin-tazobactam 100/10ug 0 0 0 67.8 96.3
Aminoglycosides Gentamycin 10ug 100 42.8 0 55.8 58.0
Tobramycin 10ug 100 57.1 0 56.5 42.8
Amikacin 30ug 0 100 0 42.1 31.4
Fluoroquinoles Ciprofloxacin 5ug 0 57.1 33.3 59.4 74.4
Folate pathway inhibitors Trimethoprim-sulfamethoxazole 1.25/23.75ug 0 100 0 79.0 68.2%
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Discussions

Infection due to Acinetobacter species is a major challenge within the health care facilities and the community in general due to their high drug resistance even to the high potent drugs such as carbapenems. In our study 102 Acinetobacter species strains were analyzed to investigate the trends of antimicrobial resistance. In the analysis we found that more than 70% of patients had taken antibiotics empirically before getting confirmed culture and antimicrobial susceptibility testing (AST) result showing Acinetobacter infection. This problem is reported in many published works elsewhere and this could contribute to the rise of exposure to multidrug resistance infection [17,18].

The observed increase in the prevalence of MDR Acinetobacter was statically significant over a period of time (chi- square, = 15.8, p value = 0.003). This might be due to the fact that the laboratory capacity to detect and conduct antimicrobial resistance testing has increased over the past years.

The overall prevalence of MDR among the isolates was 71.6% which is comparable with a report from Saudi Arabia (74%) [19] and Bosnia and Herzegovina (78.4%) [20]. However, the current finding was higher than the study conducted in Jimma which isolated the MDR Acinetobacter species from wound specimen at a rate of 57.2% [21], and in Iran at a rate of 56.7% [22]. The antimicrobial resistance profile and prevalence of hospital associated pathogens varies from place to place.

The prevalence of MDR Acinetobacter from the current study is found to be lower than a previous study done in Selected Referral Hospitals in Ethiopia involving surgical site of infection (95.7%) [23] This could be due the increasing trend of early awareness of clinicians on the treatment of Acinetobacter infection with appropriate drugs based on the microbiology laboratory identification and antimicrobials susceptibility testing results. Currently, carbapenem resistance Acinetobacter is included as target pathogen in the national AMR surveillance system in Ethiopia and this might also have contributed the decrease in the MDR status of Acinetobacter species identified in the clinical setting.

The trend analysis in antimicrobial resistance showed that in the year 2018 there was a high resistance rate for ceftriaxone (98.6%), cefepime (93.3%), ceftazidime (100%),and ciprofloxacin (74.4%) which is comparable with similar study in neighboring country Sudan which reported resistance rates as follows: ceftriaxone (95%), cefepime (92%), ceftazidime (96%) and ciprofloxacin (91%) [24].

Compared to the previous years, carbapenem resistance in Acinetobacter has increased by more than 50% by 2018 and this finding is comparable with previous studies conducted in East Africa [25] and in south East Asia [26].

The resistance to aminoglycoside from the current study was found to vary among the different agents under this antimicrobial class. While resistance to gentamycin was shown to increase from 48% to 58% from 2015 to 2018, resistance to tobramycin and amikacin was below 50%. The resistance rate reported here for tobramycin is in line with a study report from Morocco [27].

Conclusion

In conclusion, there has been an increasing trend of antimicrobials resistance in Acinetobacter species isolated from different sample source. Acinetobacter infection would remain a therapeutic challenge in our Hospitals and health care settings due to the increasing rate of Acinetobacter species with traits of MDR and resistance to high potent antimicrobial agents. Continuous surveillance and appropriate infection prevention and control program needs to be strengthened to circumvent the spread of these pathogens in the health care facilities.

Limitation of the study

Although Colistin (polymyxin E) is an antibiotic used as a last-resort for multidrug-resistant gram negative infections’ including Acinetobacter, this agent was not tested in the present study. Further molecular characterization of Acinetobacter exploring the genes responsible for MDR was not performed. All strains were not tested against carbapenem due to lack of supplies for first three years of the study.

Supporting information

S1 File

(ZIP)

Click here for additional data file. (3.2KB, zip)

Acknowledgments

We express our grateful appreciation to national clinical bacteriology and mycology reference laboratory staffs Amete mihret, Negga Asamene, Rajaha Abubeker, Surafel Fentaw,Abebe Assefa, Degefu Beyene, Dejene Shiferaw, Tesfa Addis, Yonas Mekonen,Yohanise Yitagesu,Abera Abdeta, Meseret Assefa and Etsehiwot Adamu for their technical support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

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Multi drug resistance pattern of Acinitobacter species isolated from clinical specimens refereed to Ethiopian public health institute, Ethiopia: a retrospective study.

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes the increase of resistance rates in Acinetobacter spp. strains, isolated from different types of samples, in Ethiopia. Although the authors presented interesting results, it is difficult for me to recommend this paper for publication in PLOS ONE in its present form.

1- The authors must include line numbers, as well as page numbers to help with the correction.

2- The authors should revise the English and the style of the text. There are two types of fonts along the text.

3- The authors should correct the nomenclature, in many cases, they have written Acinitobacter instead of Acinetobacter, and on several occasions, the word appears without italics.

4- The authors should homogenize the names of the antibiotics, whether with or without a capital letter, but all the same. The same applies for the percentages, between or not a parenthesis, but all the same in the same paragraph.

5- The acronym HAI has two different explanations, one in the introduction and another one in the results.

Introduction:

6- Line 7: The authors should explain the meaning of TU.

7- The first time the name of a bacteria is mentioned should be written with its complete name. The subsequent times should be written in the reducing form of its name.

8- Line 30: b-lactamase should be changed to β-lactamase.

Material and Methods

9- The section “Study period and area” is partially missing.

10- It is not clear if “Sampling technique” is a section that includes the next ones, or if the content of this section is missing.

11- The authors should explain which antimicrobial susceptibility test and how they performed it.

12- Line 12: The authors may scape the r from strains.

13- The authors should explain what statistical test they have used.

Results

14- It would be interesting if the authors could provide data relating the resistance rates of the strains with the empirical treatment received by the patients.

15- Figure 1: The authors should include the complete name of the labels.

16- Line 9: The authors should explain the meaning of CSF.

17- Line 11: Why the authors tested carbapenem susceptibility only in 37 strains, instead of in all the 102 samples?

18- Line 11: The reference to Figure 2 should be in the previous sentence.

19- The percentage of ceftriaxone for 2018 in the text does not correspond with the 2018 percentage in the table.

20- Figure 2: The authors should explain the meaning of EPHI.

21- Figures 3 and 4: The authors should include the label of the Y-axis.

22- Figures 3 and 4: I recommend the elimination of the grey background from both figures to facilitate the differentiation between the different line colours.

23- Figures 3 and 4: the meaning of the antibiotic acronyms should be explained.

Discussion

24- Line 10: What is the meaning of x^2?

25- The authors should revise the style and the content of the discussion, especially the last three paragraphs.

Reviewer #2: English needs improvement.

Absolute numbers may not be mentioned when percentage is given.

Introduction: at risk people may be described in a single sentence.

Study period: the sentence seems incomplete.

More than half of the samples were collected from two hospitals. How was the hospitals selected for this study?

Figures should be in percentage.

The references are not uniformly written.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Manas Pratim Roy

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Apr 29;16(4):e0250896. doi: 10.1371/journal.pone.0250896.r002

Author response to Decision Letter 0

11 Nov 2020

Response to reviewers

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response –Thank you editor, we have edited our manuscript based on the PLOS ONE's style requirements.

2. Thank you for stating the following financial disclosure:

[The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.].

At this time, please address the following queries:

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d. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Response – thank you editor! The authors received no specific funding for this work.” We have included this in updated cover letter.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Response –thank you we included in the method section.

4. Please ensure that you refer to Figure 4 in your text as, if accepted, production will need this reference to link the reader to the figure.

Response –thank you we referred Figure 4 in the text with some modification.

Reviewer #1: The manuscript describes the increase of resistance rates in Acinetobacter spp. strains, isolated from different types of samples, in Ethiopia. Although the authors presented interesting results, it is difficult for me to recommend this paper for publication in PLOS ONE in its present form.

1- The authors must include line numbers, as well as page numbers to help with the correction.

Response – thank you reviewer for the suggestion to follow submission guidelines we included the line numbers and page number.

2- The authors should revise the English and the style of the text. There are two types of fonts along the text.

Response – thank you we revised the English and make the fonts uniform ‘Arial’.

3- The authors should correct the nomenclature, in many cases, they have written Acinitobacter instead of Acinetobacter, and on several occasions, the word appears without italics.

Response –yes we admitted this and we use ‘find and replace’ Acinitobacter with Acinetobacter throughout the text. Thank you for this important comment.

4- The authors should homogenize the names of the antibiotics, whether with or without a capital letter, but all the same. The same applies for the percentages, between or not a parenthesis, but all the same in the same paragraph.

Response – thank you, we make it small letter for and antibiotics and make it uniform for percentages, between or not a parenthesis, throughout the texts as highlighted yellow.

5- The acronym HAI has two different explanations, one in the introduction and another one in the results.

Response –thank you reviewer we make it uniform as the acronym HAI means to show hospital acquired infection.

Introduction:

6- Line 7: The authors should explain the meaning of TU.

Response –thank you -this is a genomic species of Acinetobacter which is given by taxonomy classification in addition to species level.

7- The first time the name of a bacteria is mentioned should be written with its complete name. The subsequent times should be written in the reducing form of its name.

Response – thank you for your comment also with exception at the beginning of a sentence, we wrote it in reduced form.

8- Line 30: b-lactamase should be changed to β-lactamase.

Material and Methods

Response –we thank you. Change has been made as β-lactamase as highlighted yellow

9- The section “Study period and area” is partially missing.

Response – yes, it was missing. We filed it appropriately here after. Thank you!

10- It is not clear if “Sampling technique” is a section that includes the next ones, or if the content of this section is missing.

Response – thank you again! we mean data extraction method from archived records of the laboratory.

11- The authors should explain which antimicrobial susceptibility test and how they performed it.

Response – thank you for the comment. The test protocol for the antimicrobial susceptibility test was Kirby-Bauer disc diffusion method.

12- Line 12: The authors may scape the r from strains.

Response- thanks you! Corrected as highlighted in the text as ‘strains’

13- The authors should explain what statistical test they have used.

Descriptive ,crosstab, chi-square ,ratio, proportion,

Results

14- It would be interesting if the authors could provide data relating the resistance rates of the strains with the empirical treatment received by the patients.

Response – thank you! , we have included that in figure 2.

15- Figure 1: The authors should include the complete name of the labels.

Response- thank you we wrote the complete name

16- Line 9: The authors should explain the meaning of CSF.

Response – thank you! we mean cerebrospinal fluid as highlighted in the text yellow.

17- Line 11: Why the authors tested carbapenem susceptibility only in 37 strains, instead of in all the 102 samples?

Response –thank you! There was a shortage of the carbapenem drug during testing.

18- Line 11: The reference to Figure 2 should be in the previous sentence.

Response – thank you! We have moved fig 2 to previous

19- The percentage of ceftriaxone for 2018 in the text does not correspond with the 2018 percentage in the table.

Response –thank you! We mean 98.6% and that has been corrected

20- Figure 2: The authors should explain the meaning of EPHI.

Response –thank you we mean Ethiopian public health Institute.

21- Figures 3 and 4: The authors should include the label of the Y-axis.

Response –thank you we included the label of Y-axis.

22- Figures 3 and 4: I recommend the elimination of the grey background from both figures to facilitate the differentiation between the different line colours.

Response –thank you we eliminated the background color.

23- Figures 3 and 4: the meaning of the antibiotic acronyms should be explained.

Response –thank you, we wrote in long form.

Discussion

24- Line 10: What is the meaning of x^2?

Response –thank you x2 we mean a symbol of chi-square

25- The authors should revise the style and the content of the discussion, especially the last three paragraphs.

Response –thank you so much we have corrected the last three paragraphs

Reviewer #2: English needs improvement.

Response –thank you we have attempted to revise the English grammar and spelling errors.

Absolute numbers may not be mentioned when percentage is given.

Response –thank you we opted to use percentage and edit again in the abstract and result section

Introduction: at risk people may be described in a single sentence.

Response:Thank you for the comments. Correction has been made per the comment given.

Study period: the sentence seems incomplete.

Response .thank you, it was missed we incorporated and fill the incomplete as highlighted yellow

More than half of the samples were collected from two hospitals. How was the hospitals selected for this study?

Response –thank you, the study included all health facilities referring specimens however specimens were no growth for Acinetobacter species during culturing in some facilities. They are high load government hospitals refer sample for microbiology culture and antimicrobial susceptibility testing. This study analyzed the isolates only i.e. we excluded the no growth specimens

Figures should be in percentage.

Response .Thank you we have made change to percentage

The references are not uniformly written.

Response –thank you we revised and have written it in Vancouver style.

Attachment

Submitted filename: Response to Reviwers.docx

Click here for additional data file. (20.8KB, docx)

Decision Letter 1

Monica Cartelle Gestal

13 Jan 2021

PONE-D-20-27426R1

Multidrug resistance pattern of Acinetobacter species isolated from clinical specimens referred to Ethiopian Public Health Institute: a retrospective study.

PLOS ONE

Dear Dr. Ayenew,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please pay special attention to the comments suggested by the reviewers are they still highlight major concenrs that will improve the quality and clarity of the manuscrtpt.

==============================

Please submit your revised manuscript by Feb 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper has been clearly improve, but I would recommend the authors to revise again due to some italics, antibiotic names as well as some spaces that are missing.

Reviewer #3: the manuscript needs some attention on the following -

1. Introduction- please clearly define the multi-drug resistance of Acinetobacter species.

globally and regional data supporting the severity of multi-drug resistance of the organism (eg-mortality or

morbidity) would make the context stronger.

2. Methods- Inclusion criteria are not mentioned clearly.

3. Discussion- The result of empirical therapy is not discussed. please discuss with significance.

4. Conclusion- Please rephrase the 1st line of conclusion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Iffat Ara Ifa

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Apr 29;16(4):e0250896. doi: 10.1371/journal.pone.0250896.r004

Author response to Decision Letter 1

29 Jan 2021

Review Comments to the Author

Reviewer #1: The paper has been clearly improved, but I would recommend the authors to revise again due to some italics, antibiotic names as well as some spaces that are missing.

Response – thank you! We have made changes in our revision some italics, antibiotic names as well as some spaces missed.

Reviewer #3: the manuscript needs some attention on the following -

1. Introduction- please clearly define the multi-drug resistance of Acinetobacter species.

globally and regional data supporting the severity of multi-drug resistance of the organism (eg-mortality or

morbidity) would make the context stronger.

Response – thank you for the comments to be added and we incorporate definition of MDR –Acinetobacter species, global and regional data supporting the severity of the disease in the introduction part.

2. Methods- Inclusion criteria are not mentioned clearly.

Response – thank you we have added the inclusion criteria of the study in method part

3. Discussion- The result of empirical therapy is not discussed. please discuss with significance.

Response – thank you for the comment regarding missed discussion of empirical therapy. We have added with significance in line number 205 on page 11

4. Conclusion- Please rephrase the 1st line of conclusion.

Response – we rephrase the 1st line of conclusion in line 243 on page 13 of the manuscript.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (14.2KB, docx)

Decision Letter 2

Monica Cartelle Gestal

16 Apr 2021

Multidrug resistance pattern of Acinetobacter species isolated from clinical specimens referred to the Ethiopian Public Health Institute: 2014 to 2018 trend anaylsis

PONE-D-20-27426R2

Dear Dr. Ayenew,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: Thanks to the author for the corrections properly.

I have no other issues regarding this article. If there is any minor issue please correct it.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Iffat Ara Ifa

Acceptance letter

Monica Cartelle Gestal

21 Apr 2021

PONE-D-20-27426R2

Multidrug resistance pattern of Acinetobacter species isolated from clinical specimens referred to the Ethiopian Public Health Institute: 2014 to 2018 trend anaylsis    

Dear Dr. Ayenew:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Monica Cartelle Gestal

Academic Editor

PLOS ONE

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