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Original Article
17 (
3
); 18-25

Mutation-based fluoroquinolone resistance in carbapenem-resistant Acinetobacter baumannii and Escherichia coli isolates causing catheter-related bloodstream infections

, , ,
1Department of Microbiology and Immunology, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Nasr City, Egypt
2Department of Microbiology and Immunology, Faculty of Pharmacy, Heliopolis University, Cairo, Egypt
3Department of Microbiology and Immunology, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
4Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
5Department of Microbiology and Immunology, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Cairo, Egypt

Address for correspondence: Arwa Ramadan El Manakhly, PhD, Department of Microbiology and Immunology, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Cairo, Egypt/314 Yasmeen 6 Street, first settlement, Cairo, 11751, Egypt. Phone: 00201026995995. E-mail: arwa.ramadan@pharm.mti.edu.eg

Copyright: © International Journal of Health Sciences
Licence

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Disclaimer:
This article was originally published by Qassim Uninversity and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Objective:

We studied the presence of mutations in the chromosomal quinolone resistance-determining regions (QRDRs) of the fluoroquinolone targets gyrA and parC genes and detected the carbapenem resistance (CR) encoding genes among Acinetobacter baumannii and Escherichia coli isolates from catheter-related bloodstream infections (CRBSIs).

Methods:

The study included 39 non-duplicate isolates of A. baumannii (14/39, 35.9%) and E. coli (25/39, 64.1%) isolated from 128 confirmed CRBSIs cases. Antimicrobial susceptibility testing was performed, followed by an evaluation of biofilm formation using the tissue culture plate method. The carbapenemase encoding genes were detected by multiplex polymerase chain reaction (PCR). The mutations in QRDRs of gyrA and parC genes were determined by singleplex PCR amplification followed by DNA sequencing and BlastN analysis in the GenBank database. DNA and the translated amino acid sequences were analyzed using the Mega7 bioinformatics tool.

Results:

Multidrug-resistant (MDR) E. coli and A. baumannii isolates harbored CR encoding genes and combined gyrA and parC genes mutation. The specific substitutions observed in GyrA were Cys173Arg, Cys174Gly, Asp80Val, Tyr178ASP, Tyr84Gly, Glu85Lys, Ser172Leu, and Asp176Asn, while the specific substitutions observed in the ParC amino acid sequence were point mutation 62 Arg, Phe60Leu, Ils66Val, and Gln76Lys. Point mutation 62Arg was detected in two A. baumannii isolates, whereas Ser172Leu mutation was observed in two E. coli isolates.

Conclusion:

The presence of new single and multiple mutations in QRDR causes the emergence of MDR E. coli and A. baumannii infections in carbapenem-resistant Enterobacteriaceae in Egypt, requiring further investigation in Gram-negative bacteria.

Keywords

Acinetobacter baumannii
carbapenem resistance
catheter-related bloodstream infections
Escherichia coli
fluoroquinolone resistance

Introduction

Bloodstream infections (BSIs), specifically those caused by multidrug-resistant (MDR) bacterial pathogens, are associated with high morbidity and mortality worldwide due to the difficulty of treating with the available antimicrobial drugs.[1] One of the critical sources of BSIs is the central venous catheter (CVC). Catheter-related BSIs (CRBSIs) are laboratory-confirmed BSIs that develop within 48 h of central line placement and are not related to any infection at another body site.[2] CRBSIs remain significant healthcare-associated infections that can adversely affect patient care, causing a substantial mortality rate.[3,4]

Globally, Gram-negative bacteria (GNB), particularly Escherichia coli and Acinetobacter baumannii, have recently become prevalent in healthcare settings. Moreover, E. coli and A. baumannii are identified as leading nosocomial pathogens and among the main causes of BSIs.[5,6] These bacterial species have become increasingly multiple resistant to diverse classes of antimicrobials, particularly the most clinically used ones, including fluoroquinolones (FQs), aminoglycosides, and carbapenems.[7,8] Notably, the carbapenem-resistant A. baumannii and Enterobacteriaceae members are among the common healthcare-associated pathogens with critical priority according to the WHO global priority list of antimicrobial-resistant bacteria in 2017.[9] Indeed, infections with carbapenem-resistant Enterobacteriaceae (CRE) are a major challenge in healthcare settings and a growing concern worldwide.[8] Moreover, the resistant bacteria to carbapenems, owing to harboring the carbapenemases encoding genes such as blaOXA-48, blaNDM, and blaKPC, were found to be resistant to third-generation cephalosporins and FQs as well.[10]

FQs are bactericidal agents that are used as antimicrobial prophylaxis in immunosuppressed patients and/or primary antibacterial medication.[11] FQs target two homologous enzymes, DNA topoisomerases II (also known as DNA gyrase) and topoisomerases IV, which are essential for supercoiling bacterial DNA.[12] Both enzymes are composed of subunits encoded by gyrA and gyrB (for DNA gyrase) and parC and parE (for topoisomerase IV). The development of FQs resistance is a stepwise process resulting from the accumulation of amino acid substitutions (or mutation) in these subunits that are usually correlated with the high levels of FQs resistance.[13] These amino acid substitutions result from mutations in the quinolone resistance determining regions (QRDRs) in both gyrA and parC genes.[14] On the other hand, plasmid-mediated quinolone resistance occurs due to the plasmid-carried quinolone-resistance genes such as qyrA. These genes encode a family of proteins that protect the target enzymes from the action of quinolones.[11]

Surveillance for antimicrobial resistance is crucial to monitor the resistance trends in a developing country like Egypt. Thus, antimicrobial resistance surveillance studies are important to identify emerging MDR bacterial pathogens and resistance mechanisms, in addition to guiding for appropriate selection for empirical antimicrobial therapy and/or support the antimicrobial stewardship programs in healthcare settings.[15] Accordingly, the present study aimed to evaluate the resistance to both carbapenems and FQs among MDR E. coli and A. baumannii clinical isolates from intensive care unit (ICU) patients suffering from CRBSIs at a tertiary care hospital in Egypt. The study also aimed to investigate the presence of mutations in the chromosomal QRDRs of the fluoroquinolone resistance genes gyrA and parC and detect the carbapenem resistance (CR) encoding genes blaKPC, blaNDM and blaOXA-48.

Methods

Study patients and clinical samples

In this study, a total of 128 CRBSIs confirmed cases at a tertiary hospital located in 6th October City, Giza, Egypt, during the period from June 2016 to June 2018. All patients included in the study were ICU patients with CVC and acquired BSIs. The blood samples were collected from patients having clinical signs and symptoms of BSIs in a case of new-onset sepsis. Two sets of blood samples were drawn peripherally into BACT/ALERT blood culture bottles, incubated in BACT/ALERT system (BioMerieux, France), and monitored for 5 days. These blood samples were routinely collected and processed by the dedicated team during the medical care of ICU patients having CVC. Positive blood cultures were then recovered by streaking on MacConkey’s agar, Blood agar, and Chocolate agar plates. The plates were incubated at 37°C for 18–24 h. Gram stain reaction of the isolates was examined, and the isolates were primarily identified. The microbiological identification of the isolates was then carried out by MALDI-TOF mass spectrometry automated systems.

Determination of antimicrobial susceptibility patterns

Antimicrobial susceptibility testing was performed by the Kirby–Bauer disc diffusion method on Mueller Hinton Agar (MHA) (Oxoid, Hampshire, UK). The results were interpreted as susceptible (S), intermediate (I), or resistant (R), according to Clinical and Laboratory Standards Institute (CLSI) guidelines (30th edition).[16] The antimicrobial discs used in the current study, representing diverse classes of antimicrobials, were Ampicillin/sulbactam (10/10), cefotaxime (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), cefepime (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), and levofloxacin (5 μg), Piperacillin/tazobactam (30 μg). The isolate was verified MDR when it showed resistance to at least three different antimicrobial classes.[17]

Determination of biofilm formation

According to Ruchi et al.,[18] biofilm formation was assayed using the microtiter plate and crystal violet method. A loopful of the bacterial isolate from overnight culture was inoculated into 10 mL of trypticase soy broth containing 1% glucose and incubated overnight at 37°C. Individual wells of sterile 96 well-flat bottom polystyrene tissue culture plates (Greiner Bio-One, Germany) were filled with 200 μl of the bacterial suspension corresponding to 0.5 McFarland. The optical densities (ODs) of stained adherent bacterial films were read using a microtiter plate reader (ThermoFisher Scientific, USA) at 600 nm. The cutoff optical density (ODc) biofilm formation ability was defined as three standard deviations above the mean OD of the negative control. All isolates were classified according to their adherence capabilities into non-adherent, weak, or strong adherent based on the OD value of bacterial biofilms.[18]

The results were interpreted according to the following criteria to classify the different adherent strengths as follows: If the mean of the three repeats OD readings ≤ ODc (the mean OD plus three standard deviations of the negative control) = Non-adherent (or non-biofilm producer), ODc < OD ≤ 2 × ODc = weakly adherent (or weak biofilm producer), 2 × ODc < OD ≤ 4 × ODc = moderately adherent (or moderate biofilm producer), and if 4 × ODc < OD = strongly adherent (or strong biofilm producer). Staphylococcus aureus ATCC 29213 was used as the positive control for biofilm production.[19]

PCR-based molecular methods

DNA extraction and PCR oligonucleotide primers

For PCR detection of CR genes, total DNA was used as a template in PCR assays. Total DNA was extracted from all tested isolates using the boiling method by heating bacterial cells suspension in sterile distilled water at 100°C for 10 min, followed by removal of cellular debris by centrifugation at 14,000 rpm for 1 min. The supernatant was collected and used as template DNA for PCR amplification. For PCR amplification of QRDRs of gyrA and parC and DNA sequencing, genomic DNA was extracted from examined isolates using Gene JET Genomic DNA Purification Kit (Thermo Scientific, USA) following the manufacturer’s instructions. PCR products were purified for sequencing by QIAquick Gel Extraction Kit (QIAGEN, USA) according to the manufacturer’s protocol. The sequences of PCR oligonucleotide primers used in the current study, synthesized by Invitrogen (UK), are listed in Table 1. These primers were examined using NCBI Primer-BLAST, available at NCBI, to ensure specificity (https://www.ncbi.nlm.nih.gov/guide/data-software/).

Table 1 Nucleotide sequences of PCR oligonucleotide primers

PCR amplification and DNA sequence analysis of QRDRs of gyrA and parC genes

In the present study, the QRDRs in both gyrA and parC genes were detected by PCR in A. baumannii and E. coli isolates to determine the changes in the structure of DNA gyrase and topoisomerase IV enzymes. The genes gyrA and parC were analyzed by PCR, followed by DNA sequencing. Out of the sequenced isolates, two quinolone-sensitive isolates were included as a control.

The QRDRs of gyrA and parC genes were amplified by single-plex PCR. The PCR reaction mixtures were prepared in total volumes of 20 μl. Each reaction contained 2 μl of template DNA, 1 μl of each primer and 10 μL of GoTaq® Green Master 2× Ready Mix (Promega, USA), then the volume was completed to 20 μL by adding 6 μL of nuclease-free water. The PCR amplification program was as follows: Initial denaturation for 5 min at 95°C, then 30 cycles of denaturing at 95°C for 30 s, annealing for 30 s at 47°C for parC gene and 53°C for gyrA gene, and extension at 72°C for 45 s, followed by a final extension at 72°C for 7 min. The PCR-amplified QRDRs were subjected to DNA sequencing using the technology of Sanger sequencing using Applied Biosystems 3500 Genetic Analyzer at Clinilab, Cairo, Egypt. The obtained DNA sequences and their predicted amino acid sequences were analyzed using online bioinformatics tools, including BLAST analyses tools (blastn and blastp) (http:/www.ncbi.nlm.nih.gov/BLAST/). The multiple sequence alignment tool (ClustalW) and Mega software version 7.0.26 were used.

Multiplex-PCR for detection of carbapenemases encoding genes

The carbapenemases encoding genes blaKPC, blaNDM, and blaOXA-48 were investigated using a multiplex PCR assay previously described by Poirel et al.[22]

Statistical analysis

Data are presented as numbers and percentages for categorical variables, and biofilm data are expressed as the mean ± standard deviation (SD).

Results

Identification and frequencies of A. baumannii and E. coli isolates recovered from different clinical samples

A total of 39 non-duplicate MDR bacterial clinical isolates of A. baumannii (14/39, 35.9% isolates) and E. coli isolates (25/39, 64.1% isolates) were recovered from the 128 blood samples included in the current study. The bacterial isolates recovered from the blood samples were of other bacterial species that were not targeted in the present study.

Antimicrobial susceptibility profiles of A. baumannii and E. coli isolates

Overall, there were high resistance levels among A. baumannii and E. coli isolates to the tested antimicrobial agents. Antimicrobial resistance profiles revealed that all (100%) A. baumannii and E. coli isolates were resistant to ampicillin, ampicillin/sulbactam, and amoxicillin/clavulanic acid. All A. baumannii isolates were resistant to ciprofloxacin and 92.86% were resistant to levofloxacin. E. coli isolates also showed resistance rates to ciprofloxacin and levofloxacin of 76% (19/25) and 36% (9/25), respectively. Regarding carbapenems, 100% (14/14) of A. baumannii isolates were resistant to each imipenem and meropenem, while 44% (11/25) and 64% (16/25) of E. coli isolates were resistant to imipenem and meropenem, respectively [Table 2].

Table 2 Antimicrobial resistance profiles of A. baumannii and E. coli isolates

Biofilm formation ability among isolates

The tissue culture plate method performed to assess the biofilm formation ability among A. baumannii quantitatively and E. coli isolates revealed that 21.43% (3/14) of A. baumannii isolates and 25% (3/12) of E. coli isolates are the only microorganisms showed the ability to produce biofilm. All the isolates forming biofilm were described as moderate biofilm formation.

Detection of common carbapenemase encoding genes

In the present study, the most predominant carbapenemase encoding gene among E. coli and A. baumannii was blaNDM, as it was detected in 44% (4/25) and 50% (7/14), respectively. Collectively, blaNDM was the predominant carbapenemase gene in 46.15% (18/39) followed by blaKPC was detected in 17.95% (7/39) and blaOXA48 was detected in 2.26% (1/39).

Mutations in QRDRs of gyrA and parC genes in FQs-resistant isolates

The QRDRs in both gyrA and parC genes in A. baumannii and E. coli isolates were subjected to PCR amplification [Figure 1], followed by DNA sequencing and BLAST analyses. It was found that two isolates showed no mutation in the QRDR, while A. baumannii showed combined gyrA and parC mutations. A combined substitution was observed in all E. coli and A. baumannii isolates [Figure 2] that showed gene mutations. The specific substitutions observed in GyrA were Cys173Arg, Cys174Gly, Asp80Val, Tyr178ASP, Tyr84Gly, Glu85Lys, Ser172Leu, and Asp176Asn. While the specific substitutions observed in ParC were point mutation 62Arg, Phe60Leu, Ils66Val, and Gln76Lys. Point mutation 62 Arg was observed in two A. baumannii isolates, whereas Ser172Leu mutation was observed in two E. coli isolates as shown in Table 3.

Single plex polymerase chain reaction for amplification of gyrA and parC
Figure 1
Single plex polymerase chain reaction for amplification of gyrA and parC
Example of the mutation and substitution of parC found in Acinetobacter baumannii isolate
Figure 2
Example of the mutation and substitution of parC found in Acinetobacter baumannii isolate
Table 3 Mutations in gyrA and parC in A. baumannii and E. coli:

Discussion

E. coli and A. baumannii bacterial species have recently been identified as leading nosocomial pathogens and among the main causes of BSIs.[5,6] According to National Healthcare Safety Network (NHSN) data, E. coli and Acinetobacter spp. are considered the most common etiologies for CLABSI.[23-25] In addition, mortality rates associated with invasive A. baumannii infection are relatively high, especially for carbapenem-resistant cases. The crude mortality for carbapenem-resistant A. baumannii infections ranges from 16% to 76%, compared to 5–53% for carbapenem-susceptible infections.[26] Moreover, attributable mortality of 70% has been reported for BSIs due to imipenem-resistant A. baumannii, compared with 24.5% for imipenem-susceptible A. baumannii in Taiwan.[27] Consequently, the therapeutic options are limited, particularly in critically ill patients. Antimicrobial resistance patterns differ considerably from country to country or among hospitals in the same country and within the same hospital over time.[28,29] Thus, the regular surveillance of nosocomial pathogens for prevalence and antimicrobial resistance outlines is warranted for appropriate empirical antimicrobial therapy. Accordingly, in this study, nosocomial E. coli and A. baumannii blood isolates from confirmed CRBSI cases were screened for their antimicrobial susceptibility patterns. In addition, FQs-resistant isolates were investigated for the quinolone resistance mechanism through mutations in QRDRs of the chromosomal FQs target genes gyrA and parC.[30]

Compared to the conventional microbiological identification methods, MALDI-TOF MS showed a precise identification rate of 100% of the target species and reduced the typical turn-around time with no loss of accuracy, providing a fast and accurate method for the identification of these bacteria, particularly in crowded health-care settings.[31,32]

The presence of indwelling devices can cause serious health-care problems, specifically with the production of biofilm that allows bacteria to colonize the indwelling devices and form a shield to protect microbes against antimicrobial agents.[33] Indeed, the previous studies revealed that biofilm formation is associated with the resistance of microorganisms, such as E. coli, toward antimicrobial agents, and biofilm formation increases the incidence of healthcare-associated infections, especially in CRBSIs.[34,35] The tissue culture plate method was used in this study to examine whether bacterial isolates can form biofilm. Based on the results of evaluating biofilm formation ability, six isolates showed a moderate ability to form a biofilm. On the other hand, previous studies showed the higher rates of biofilm production among nosocomial isolates than our rates which could be correlated with the duration of hospitalization and/or prior antibiotic administration.[18,19,36]

The present results agreed with several studies that reported high resistance patterns of GNB to β-lactams in Egypt and worldwide.[27,37-40] On the other hand, our bacterial isolates were 100% sensitive to colistin and polymixin, which appeared to be the most effective antimicrobial agents against E. coli and A. baumannii isolates. Several studies revealed that FQs are typically used in combination with other antimicrobial agents to treat carbapenem-resistant pathogens.[41-43] Also, the results of the present study agreed with recent Egyptian studies that identified E. coli and carbapenem-resistant Acinetobacter spp. isolates as MDR organisms resistant to at least one antimicrobial agent in three or more different antimicrobial classes. Therefore, those pathogens have become target pathogens in national Egyptian Antimicrobial resistance in Egypt to decrease the MDR status identified in the clinical settings.[15,17,31,44]

Regarding E. coli, all (25/25, 100%) isolates were resistant to each ampicillin, ampicillin/sulbactam, aztreonam, ceftazidime, and ceftriaxone. Similar findings were reported by another previous study from Lahore by Sabir et al. (2014), who stated that 100% of the E. coli isolates were resistant to penicillin, in addition to 62.6%, 89.50% and 73.80% of isolates were resistant to amoxicillin/clavulanate, cefotaxime, and ceftazidime, respectively.[36] In the present study, E. coli isolates showed varied resistance patterns to the other antimicrobial agents; 64% (16/25) and 36% (9/25) of the isolates showed resistance to gentamicin and ciprofloxacin, respectively. Our findings agree with the Mohammadi et al. study in which E. coli isolates showed resistance to ciprofloxacin, gentamicin, piperacillin/tazobactam and amikacin with frequencies of 60%, 31.66%, 33.33%, and 11.66%, respectively.[37]

The current study revealed that the resistance rate among A. baumannii isolates was 92.86% to both imipenem and meropenem. This record was in agreement with a previous study from Egypt, which reported 74% resistance to imipenem among carbapenem-resistance isolates.[35] Notably, this study’s rates of antimicrobial resistance are much higher than previous reports from the same hospital and/or other hospitals in Egypt.[39,40]

There is an increasing rate of resistance to FQs among Gram-negative isolates worldwide.[28] The present study findings agreed with a recent study by Lo et al., who reported resistance frequencies of 70.9% and 65.3% to ciprofloxacin and levofloxacin, respectively.[45] Consistent with our study, Yang et al. surveyed 130 hospitals in China and showed that the median resistance rate of A. baumannii to FQs was 59.3%, and the median resistance rate of E. coli to FQ was 61.67%.[46]

Changes in the structure of the FQs target enzymes DNA gyrase and DNA topoisomerase IV are important mechanisms in conferring resistance to FQs in GNB. In E. coli, three or four mutations in both gyrA and parC genes were found necessary to obtain a high level of resistance to FQs, whilst double mutations at positions 83 (Ser83) of gyrA and 80 (Ser80) of parC led to a moderate level of resistance to FQs.[28] In the present study, sixteen representative isolates were sequenced then the obtained sequences were subjected to bioinformatic analysis using NCBI BlastN function against the GenBank database to detect the mutation in QRDRs of gyrA and parC genes. The investigated A. baumannii isolates showed combined mutations in both gyrA and parC encoding genes that explained the high resistance rates among the tested isolates. On the other hand, one isolate of E. coli showed a combination of three mutations, and the other three E. coli isolates showed only one mutation. Our results agree with Ardebili et al.[28] who reported that three or four mutations in the gyrA gene are necessary to obtain a high level of resistance to ciprofloxacin in E. coli. On the other hand, double mutations of parC cause only moderate-level resistance.[28] Our results were also in agreement with recent studies,[47-49] which reported that combined mutation of parC and gyrA was associated with resistance and suggested that the presence of gyrA and parC mutations at codon 83 and codon 80 with substitution of serine with leucine in gyrA and serine with leucine in parC were the most common mutations in A. baumannii. In addition, mutation at position 80 in parC was observed in 93% of isolates in A. baumannii in Iran, and all of which were resistant to ciprofloxacin and levofloxacin.[41] An earlier study by Vila et al.[50] reported different types of gene mutations, such as Ala84Pro or Gly81Val, in ciprofloxacin-resistant isolates. However, in the present study, other mutations were detected and associated with a high level of resistance to quinolones recorded in the present study. The specific substitutions observed in gyrA were Cys173Arg, Cys174 Gly, Asp80Val, Tyr178ASP, Tyr84Gly, Glu85Lys, Ser172Leu, and Asp176Asn. While the specific substitutions observed in parC were point mutation 62 Arg, Phe60Leu, Ils66Val, and Gln76Lys. Point mutation 62Arg was observed in two A. baumannii isolates, whereas Ser172Leu mutation was observed in two E. coli isolates.

The increased consumption of carbapenems may lead to major selection pressure, which would enrich the preexisting mutants of resistant A. baumannii and E. coli and result in the development of CR and other antimicrobial agents such as FQs.[51] The prevalence of the blaNDM gene was 46.15% among carbapenem-resistant isolates and represented the predominant carbapenem-resistance encoding gene. The distribution of blaNDM gene in this study was comparable to another Egyptian study that described the blaNDM as the most prevalent carbapenemase resistance encoding gene in a university hospital in Egypt.[52] In the present study, the KPC encoding gene was detected among the tested isolates with a percentage of 17.95%. This is relatively in agreement with other studies that stated KPC carriage by GNB is not the main cause of CR in the Middle East and Egypt.[53,54] Regarding blaOXA-48, only 2.56% of carbapenem-resistant isolates harbored this gene. In agreement with our study, other studies detected only 4.6% and 9.7% of blaOXA-48 in Egypt.[52,55] Earlier surveillance study of carbapenem-resistant GNB in a cancer hospital in Egypt, only three isolates harbored blaOXA-48.[55] In contrast to our results, Asem et al.[54] reported a higher number of isolates carrying blaOXA-48 which may indicate the rapid dissemination of blaOXA-48 genes. This marked increase in the rates of antimicrobial resistance and high dissemination of resistance encoding genes and/or mutation could be explained by the lack of a national antimicrobial stewardship program, misuse and overuse of antibiotics in human, animal, and plant care, and the inconsistency of implementation of national infection control guidelines.[15,31,56]

Conclusion

A. baumannii and E. coli isolates showed the high frequencies of carriage of carbapenem-resistance encoding genes and the coexisting of several mutations within QRDR regions of the gyrA and parC genes are expected to contribute to high-level fluoroquinolone resistance among the tested isolates. The accumulation of triple mutations in the QRDR of the gyrA and parC genes leads to minimal therapeutic options and calls for further investigation of the mutation in these genes in addition to strict infection control policy and an antimicrobial stewardship program implementation in Egyptian hospitals.

Authors Declaration Statements

Ethics approval and consent to participate

The study protocol was approved by the Research Ethics Committee of Cairo University Medical School in accordance with the Declaration of Helsinki (Ethical approval number: N-13-2020).

Availability of data and material

The data that support the findings of this study are available from the corresponding author on reasonable request.

Competing interests

All the authors declared that there is no conflict of interest. All authors declared that the work is original and does not infringe the copyright or other party’s property rights.

All authors have read and approved this submission and have given appropriate credit to everyone who participated in this work.

Funding statement

This research is self-funded and not supported by funding sources, grants, or not-for-profit sectors.

Authors Contribution Statement

The following was the contribution, according to the authors: Assoc. Prof. Mahmoud M. Tawfick supervised the practical work and writing and revising of the manuscript. Prof. Abeer Khairy provided: critical feedback and analyzed the practical work. Prof. Amani El-Kholy provided critical feedback, and led the practical work. Dr. Arwa Ramadan conducted the practical work, and the data analysis and wrote the manuscript.

Acknowledgments

NA.

References

  1. , , , , , , . Bloodstream infections caused by multidrug-resistant gram-negative bacteria:Epidemiological, clinical and microbiological features. BMC Infect Dis. 2019;19:609.
    [Google Scholar]
  2. , , , , . Establishing catheter-related bloodstream infection surveillance to drive improvement. J Infect Prev. 2018;19:160-6.
    [Google Scholar]
  3. , , , , , , . Changes in prevalence of health care-associated infections in U. S. hospitals. N Engl J Med. 2018;379:1732-44.
    [Google Scholar]
  4. , , , , , , . Inpatient costs, mortality and 30-day re-admission in patients with central-line-associated bloodstream infections. Clin Microbiol Infect. 2014;20:O318-24.
    [Google Scholar]
  5. , , . Carbapenem resistance:A review. Med Sci (Basel). 2017;6:1.
    [Google Scholar]
  6. , . Acinetobacter spp as nosocomial pathogens:Epidemiology and resistance features. Saudi J Biol Sci. 2018;25:586-96.
    [Google Scholar]
  7. , . Resistance in gram-negative bacteria:Enterobacteriaceae . Am J Med. 2006;119(6 Suppl 1):S20-8. discussion S62-70
    [Google Scholar]
  8. , , , , , , . KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011:Massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units. Gut Pathog. 2012;4:7.
    [Google Scholar]
  9. , , , , , . WHO pathogens priority list working group. Discovery, research, and development of new antibiotics:The WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet. Infectious Diseases. 2018;18(3):318-327.
    [Google Scholar]
  10. , , , , , , . Treatment options for carbapenem-resistant Gram-negative infections. Dtsch Arztebl Int. 2018;115:345-52.
    [Google Scholar]
  11. , , , , , , . Multiple mechanisms contributing to ciprofloxacin resistance among gram negative bacteria causing infections to cancer patients. Sci Rep. 2018;8:12268.
    [Google Scholar]
  12. , . Mechanisms of quinolone action and microbial response. J Antimicrob Chemother. 2003;51(Suppl 1):29-35.
    [Google Scholar]
  13. , . Mechanisms of resistance to quinolones:Target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother. 2003;51:1109-17.
    [Google Scholar]
  14. , , , . Antibacterial-resistant Pseudomonas aeruginosa:Clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev. 2009;22:582-610.
    [Google Scholar]
  15. , , , , , . Molecular characterization of multidrug-resistant gram-negative pathogens in three tertiary hospitals in Cairo, Egypt. Eur J Clin Microbiol Infect Dis. 2020;39:987-92.
    [Google Scholar]
  16. . Performance Standard for Antimicrobial Susceptibility Testing (30th ed). Wayne, PA: Clinical and Laboratory Standard Institute; . CLSI Supplement M100
  17. , , , , , , . Multidrug-resistant, extensively drug-resistant and pandrug-resistance bacteria:An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18:268-81.
    [Google Scholar]
  18. , , , . Comparison of phenotypic methods for the detection of biofilm production in uro-pathogens in a tertiary care hospital in India. Int J Curr Microbiol App Sci. 2015;4:840-9.
    [Google Scholar]
  19. , , , , , , . Correlation between biofilm-formation and the antibiotic resistant phenotype in Staphylococcus aureus isolates:A laboratory-based study in Hungary and a review of the literature. Infect Drug Resist. 2021;14:1155-68.
    [Google Scholar]
  20. , , , , , . Correlation of quinolone resistance levels and differences in basal and quinolone-induced expression from three qnrA-containing plasmids. Clin Microbiol Infect. 2006;12:440-5.
    [Google Scholar]
  21. , , , , , , . In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid. J Antimicrob Chemother. 2006;58:1054-7.
    [Google Scholar]
  22. , , , , . Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011;70:119-23.
    [Google Scholar]
  23. , , , , , . Central line-associated blood stream infections:Characteristics and risk factors for mortality over a 5.5-year period. Turk J Med Sci. 2017;47:646-52.
    [Google Scholar]
  24. , , , , , . Healthcare-associated infections studies project:An American Journal of Infection Control and National Healthcare Safety Network data quality collaboration:Location mapping. Am J Infect Control. 2018;46:577-8.
    [Google Scholar]
  25. , , , . Central line associated blood stream infections. In: In:StatPearls. Treasure Island: StatPearls Publishing; . p. :28613641.
    [Google Scholar]
  26. , , , , , , . Carbapenem resistance and mortality in patients with Acinetobacter baumannii infection:Systematic review and meta-analysis. Clin Microbiol Infect. 2013;20:416-23.
    [Google Scholar]
  27. , , , , , . Risk factors and outcome analysis of Acinetobacter baumannii complex bacteremia in critical patients. Crit Care Med. 2014;42:1081-8.
    [Google Scholar]
  28. , , , , . Association between mutations in gyrA and parC genes of Acinetobacter baumannii clinical isolates and ciprofloxacin resistance. Iran J Basic Med Sci. 2015;18:623-6.
    [Google Scholar]
  29. , , , , , , . Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clin Microbiol. 2010;48:1169-75.
    [Google Scholar]
  30. , , , , , . Antimicrobial susceptibility patterns of an emerging multidrug resistant nosocomial pathogen:Acinetobacter baumannii . Malays J Med Sci. 2018;25:129-34.
    [Google Scholar]
  31. , , , , . Carbapenem-resistant gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt. Eur J Clin Microbiol Infect Dis. 2018;37:1647-52.
    [Google Scholar]
  32. , , , , , , . Assessment of reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry for bacterial and yeast identification. J Clin Microbiol. 2015;53:2349-52.
    [Google Scholar]
  33. , , , , . Bacteremia due to ESKAPE pathogens:An emerging problem in cancer patients. J Egypt Natl Canc Inst. 2016;28:157-62.
    [Google Scholar]
  34. , , , . Antibiotic-resistant gram-negative bacterial infections in patients with cancer. Clin Infect Dis. 2014;59(Suppl 5):S335-9.
    [Google Scholar]
  35. , , , . The frequency and antimicrobial resistance patterns of nosocomial pathogens recovered from cancer patients and hospital environments. Asian Pac J Trop Biomed. 2015;5:1055-9.
    [Google Scholar]
  36. , , , , , , . Isolation and antibiotic susceptibility of E.coli from urinary tract infections in a tertiary care hospital. Pak J Med Sci. 2014;30:389-92.
    [Google Scholar]
  37. , , . Antimicrobial resistance pattern of gram-negative bacilli isolated from patients at ICUs of Army hospitals in Iran. Iran J Microbiol. 2011;3:26-30.
    [Google Scholar]
  38. , , , , . Emergence of carbapenem-resistant Acinetobacter baumannii harboring the OXA-23 carbapenemase in intensive care units of Egyptian hospitals. Int J Infect Dis. 2013;17:e1252-4.
    [Google Scholar]
  39. , , , , , . Antimicrobial resistance in Cairo, Egypt 1999-2000:A survey of five hospitals. J Antimicrob Chemother. 2003;51:625-30.
    [Google Scholar]
  40. , , , , , , . Device-associated nosocomial infection rates in intensive care units at Cairo university hospitals:First step toward initiating surveillance programs in a resource-limited country. Am J Infect Control. 2012;40:e216-20.
    [Google Scholar]
  41. , , , , . In vitro activity of delafloxacin against contemporary bacterial pathogens from the United States and Europe, 2014. Antimicrob Agents Chemother. 2017;61:e02609-16.
    [Google Scholar]
  42. , , , , , . Plazomicin:A novel aminoglycoside for the treatment of resistant gram-negative bacterial infections. Drugs. 2019;79:243-69.
    [Google Scholar]
  43. , , , , , , . Multidrug resistant Acinetobacter baumannii:Resistance by any other name would still be hard to treat. Curr Infect Dis Rep. 2019;21:46.
    [Google Scholar]
  44. , , , , , . The emergence of carbapenemase blaNDM genotype among carbapenem-resistant Enterobacteriaceae isolates from Egyptian cancer patients. Eur J Clin Microbiol Infect Dis. 2020;39:1251-9.
    [Google Scholar]
  45. , , , , , , . Fluoroquinolone therapy for bloodstream infections caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae. J Microbiol Immunol Infect. 2017;50:355-61.
    [Google Scholar]
  46. , , , , , , . Association between the rate of fluoroquinolones-resistant gram-negative bacteria and antibiotic consumption from China based on 145 tertiary hospitals data in 2014. BMC Infect Dis. 2020;20:269.
    [Google Scholar]
  47. , , , , . Mechanism of action of and resistance to quinolones. Microb Biotechnol. 2009;2:40-61.
    [Google Scholar]
  48. , , , , . Detection of quinolone-resistance mutations of parC gene in clinical isolates of Acinetobacter baumannii in Iran. J Res Med Sci. 2014;19:567-70.
    [Google Scholar]
  49. , , , . Molecular study of quinolone resistance determining regions of gyrA gene and parC gene s in clinical isolates of Acinetobacter baumannii resistant to fluoroquinolone. Open Microbiol J. 2018;12:116-22.
    [Google Scholar]
  50. , , , , , . Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrob Agents Chemother. 1995;39:1201-3.
    [Google Scholar]
  51. , , , , , , . Correlation between carbapenem consumption and antimicrobial resistance rates of Acinetobacter baumannii in a university-affiliated hospital in China. J Clin Pharmacol. 2013;53:96-102.
    [Google Scholar]
  52. , , , . Risk factors, phenotypic and genotypic characterization of carpabenem resistant Enterobacteriaceae in Tanta university hospitals, Egypt. Int J Infect Control. 2016;12:1-11.
    [Google Scholar]
  53. , , , , , , . The emergence of OXA-48- and NDM-1-positive Klebsiella pneumoniae in Riyadh, Saudi Arabia. Int J Infect Dis. 2013;17:e1130-3.
    [Google Scholar]
  54. , , , , , , . Emergence of gram-negative bacilli with Concomitant blaNDM-1 and blaOXA-48-like genes in Egypt. Am J Intern Med. 2017;5:1-6.
    [Google Scholar]
  55. , , , , , . Prevalence and characterization of carbapenem-resistant Enterobacteriaceae isolated from Mulago national referral hospital, Uganda. PLoS One. 2015;10:e0135745.
    [Google Scholar]
  56. , , , , . Occurrence of OXA-48 and VIM-1 carbapenemase-producing Enterobacteriaceae in Egypt. Int J Antimicrob Agents. 2013;41:90-1.
    [Google Scholar]

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