Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: insights from a tertiary hospital in Southern Thailand

ABSTRACT Broad-spectrum ampicillin-resistant and third-generation cephalosporin-resistant Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae that have pathological features in humans, have become a global concern. This study aimed to investigate the prevalence, antimicrobial susceptibility, and molecular genetic features of extended-spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumoniae isolates in Southern Thailand. Between January and August 2021, samples (n = 199) were collected from a tertiary care hospital in Southern Thailand. ESBL and AmpC-lactamase genes were identified using multiplex polymerase chain reaction (PCR). The genetic relationship between ESBL-producing E. coli and K. pneumoniae was determined using the enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction. ESBL-producing E. coli and K. pneumoniae isolates were mostly collected from catheter urine samples of infected female patients. The ESBL production prevalence was highest in the medical wards (n = 75, 37.7%), followed by that in surgical wards (n = 64, 32.2%) and operating rooms (n = 19, 9.5%). Antimicrobial susceptibility analysis revealed that all isolates were resistant to ampicillin, cefotaxime, ceftazidime, ceftriaxone, and cefuroxime; 79.4% were resistant to ciprofloxacin; and 64.3% were resistant to trimethoprim-sulfamethoxazole. In ESBL-producing K. pneumoniae and E. coli, blaTEM (n = 57, 72.2%) and blaCTX-M (n = 61, 50.8%) genes were prominent; however, no blaVEB, blaGES, or blaPER were found in any of these isolates. Furthermore, only ESBL-producing K. pneumoniae had co-harbored blaTEM and blaSHV genes at 11.6%. The ERIC-PCR pattern of multidrug-resistant ESBL-producing strains demonstrated that the isolates were clonally related (95%). Notably, the presence of multidrug-resistant and extremely resistant ESBL producers was 83.4% and 16.6%, respectively. This study highlights the presence of blaTEM, blaCTX-M, and co-harbored genes in ESBL-producing bacterial isolates from hospitalized patients, which are associated with considerable resistance to beta-lactamase and third-generation cephalosporins. IMPORTANCE We advocate for evidence-based guidelines and antimicrobial stewardship programs to encourage rational and appropriate antibiotic use, ultimately reducing the selection pressure for drug-resistant bacteria and lowering the likelihood of ESBL-producing bacterial infections.


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The authors in this paper surveyed 199 E. coli and K. pneumoniae 199 isolates from patients for their resistance to various antibiotics and the presence of antibiotic resistance genes.
Notes: Introduction lines 82-86.All the different classes of beta-lactamases are listed by their acronyms, eg.OXA, SHV etc.But what is not listed is what they stand for, ie.Oxacillinase (OXA).What is not listed is the class B type, metallo beta-lactamases in the introduction.The authors should note that beta-lactamases have four classes, and OXA is class D. This needs to be made clearer.
Results line 236-7.The sentence reads as though all isolates contained all three bands, but I'm sure the authors mean that all 199 isolates were positive for at least one band, not all three.Otherwise, the rest of the analysis does not make sense.
In the materials and methods section, it is mentioned that "ESBL isolates with a similarity coefficient {greater than or equal to} 85% were considered the same genotype (20)."How many of the 199 isolates were considered the same genotype?Discussion line 263-265: Place the reference next to the reported percentage, rather than at the end of the sentence.
It's good that the authors acknowledge the limitations of the study.They should also note that there's a strong chance that many of the isolates are clonal, hence the fact that the ERIC-PCR result shows that nearly all the isolates are from group A (and 100% of the K. pneumoniae ones were).The strong possibility that many of the isolates are clonal needs to be mentioned here (lines 302-316).

Figures:
The labelling for figure one needs to be improved.State in the legend which lanes have which samples and remove the written text from the gel as it is very confusing.Also state what size each band should be in the figure legend.
Figure 2: It is very hard to read figure 2. It looks like there is text at the end of each line on the dendogram, but it is impossible to read it.Either remove the text or find a way to identify the isolates in an easier way (ie.Different colours for E. coli or K. pneumoniae).Also, could a gel of the ERIC-PCR be included in the figure ?For tables 4 and 5, state which test you are using to get a p-value (student t-test, odds ratio?)Overall, the paper reads well and the authors do analyse the data they have presented in a clear and concise manner.The figures need to be improved for publication quality (see above).It is good to know that most of the isolates are carbapenem sensitive, probably owing to the lack of blaPER alleles identified.
One other point, the authors state that they sequenced the alleles.But I did not read any analysis on whether all isolates had the same type of CTX-M allele or TEM type.Were they identical or were different allele types of the resistance genes found?A more detailed discussion on that point should be included.
Reviewer #2 (Comments for the Author): Abstract: Line 30-32: The description provided is unclear; kindly stick to ESBL-EC and KP or AMPC-EC and KP.Also, the statement seems ambiguous; could you please clarify or rewrite it for better clarity.Furthermore, I noticed that you later introduced AMPC genes or AMPC beta-lactamase genes alongside ESBL genes.However, I am unsure why you would describe them as AmpC-lactamase genes, as I do not believe this is a description that exists.
A similar issue arises in line 56: "which are associated with considerable resistance to beta-lactamase and third generation cephalosporins".Is this an oversight in writing, or do the authors need to review the facts before writing them up?Resistance to beta-lactamase, really?Keywords: There are too many keywords and many of them are unrequired.I question whether "ERIC PCR" should be included as a keyword for this paper, for example?Introduction: Could the authors ensure that the introduction section includes sufficient citations for the statements where they are currently missing?Line 72-74: Once more, referencing my earlier statement, I would like to address a few concerns: based on my statement earlier.Is this: "Extended-spectrum β-lactamases (ESBLs) that produce E. coli and K. pneumoniae are major causes of childhood infections and pose significant challenges, such as........." an oversight in writing, or do the authors need to review the facts before writing them up?
Additionally, why start sentences with abbreviations (Check lines 72 and 82).Also; Lines 72-81 are so uncoordinated.The paragraph is so painful to read.Line 82: Really now: "ESBLs are caused by TEM-1, TEM-2, and SHV-1 β-lactamase mutations".How is this possible?Again: ESBL-producing Enterobacteriaceae resistance to antimicrobial agents, especially E. coli and K. pneumoniae, poses a substantial issue in nosocomial infections and community settings.What is this supposed to mean?Reviewer #4 (Comments for the Author): Overview of manuscript: In this paper, the authors examined bacteria previously isolated from hospital patient samples (from a hospital in Southern Thailand) to determine the prevalence of ESBL strains in this locality.They also analyzed their resistance to a few non-B-lactam drugs and used PCR to determine fingerprints for the strains as well as which resistance genes were present.One limitation of the study was that all samples were from the same hospital and may not be representative of the region.However, this study does provide some information as to the prevalence ESBL strains in this area.

Specific comments:
Line 72: rephrase -ESBLs do not produce E. coli and K. pnuemoniae Line 82: provide a brief description/explanation of these 3 mutations Lines 161 and 178: the "buffer" needs to be defined, meaning its composition needs to be reported Lines 178-179: provide the final concentrations of the components (like was done in lines 161-163) rather than volumes and stock concentrations; in addition, it is implied that "empty space" in the reaction is filled with DI water, so this does not need to be stated Lines 207-232, 286, and potentially elsewhere: Many of the % reported in the manuscript do not match the % reported in the accompanying table; some appear to be variations in rounding and/or rounding errors.However, all of these discrepancies should be corrected, and if any rounding is done, it should be consistent (i.e., to the same decimal point) throughout the manuscript.In addition, in Line 207, these two % add to greater than 100%, so a math error has been made and should be corrected.Lines 208-209: "maximum number of ESBL-producing bacteria" -it is unclear what exactly is meant by this.Does this mean that all strains identified could be accounted for the in 60+ population and repeats of them were found in younger populations?Does it mean that the greatest variety of strains from a single patient was found in someone over 60?In addition, it is unclear why 60 was chosen as the cut-off when this is not an age cut-off in the table of data provided.Line 211: "Of these isolates" is vague.Does this refer to isolates from catheter urine?Lines 211-212: 3 figures for % are provided for only 2 things, so there appears to be an extra number inserted Lines 216-217 seem to be a repeat of what was stated a few sentences earlier Lines 223-225: it is not clear why parentheses are used here Line 224: ciprofloxacin data is not reported in the table.This should be corrected.Lines 226-227: I don't follow how the prevalence of ESBL-SUSCEPTIBLE isolates was high.In addition, the range of rates for the "other antimicrobials" should be included so readers can judge the comparison for themselves.Line 232: PDR-PA strains are not mentioned anywhere in Table 5, so there is no reason to reference this table here.Line 235 and elsewhere: blaCTX-M seems to have about 3 different names throughout the manuscript, tables, and figures -be consistent throughout.If these are referring to different variations, then that needs to be specified and the differences (and their significance) explained.Line 247: briefly explain ERIC PCR either in the Introduction or here as all readers who are interested in resistance may not be versed in this technique Line 248: "therefore" should be replaced with "and" Line 251: explain what is meant by "unambiguously genotyped" Lines 253-254: explain how this conclusion was reached Figure 1: whatever is at the ends of the dendrogram branches is not legible; in addition, the figure caption needs to explain what A, B, and U are.It would also be informative for many readers to include an ERIC gel from some small portion of these isolates.Figure 2: label ladder bands with bp sizes; remove line around the text boxes on the gel; it is not clear what the labels on the gel correspond to, especially because there seems to be many more band sizes than labels; in the caption, for Lanes 1-6, there are 5 things listed corresponding to the 6 lanes, so it is not clear what corresponds to which lanes; presumably lanes 4 and 5 are the lanes showing "co-harboring" yet the bands in those lanes are not the same size as bands in any other lanes despite these supposedly being present singly in other isolates; similarly, there are 3 things listed for lanes 7-13, so it is again not clear what corresponds to each lane.Table 2: it is not clear what the p-values correspond to/measure Table 2: in light of lines 259-260, the number of specimens/samples in each category should be stated in the table.This will also help readers see how often multiple isolates were found in a single sample.Tables 4 and 5 Results line 236-7.The sentence reads as though all isolates contained all three bands, but I'm sure the authors mean that all 199 isolates were positive for at least one band, not all three.Otherwise, the rest of the analysis does not make sense.
In the materials and methods section, it is mentioned that "ESBL isolates with a similarity coefficient ≥ 85% were considered the same genotype (20)."How many of the 199 isolates were considered the same genotype?Discussion line 263-265: Place the reference next to the reported percentage, rather than at the end of the sentence.
It's good that the authors acknowledge the limitations of the study.They should also note that there's a strong chance that many of the isolates are clonal, hence the fact that the ERIC-PCR result shows that nearly all the isolates are from group A (and 100% of the K. pneumoniae ones were).The strong possibility that many of the isolates are clonal needs to be mentioned here (lines 302-316).

Figures:
The labelling  It looks like there is text at the end of each line on the dendogram, but it is impossible to read it.Either remove the text or find a way to identify the isolates in an easier way (ie.Different colours for E. coli or K. pneumoniae).Also, could a gel of the ERIC-PCR be included in the figure ?For tables 4 and 5, state which test you are using to get a p-value (student t-test, odds ratio?)Overall, the paper reads well and the authors do analyse the data they have presented in a clear and concise manner.The figures need to be improved for publication quality (see above).It is good to know that most of the isolates are carbapenem sensitive, probably owing to the lack of bla PER alleles identified.
One other point, the authors state that they sequenced the alleles.But I did not read any analysis on whether all isolates had the same type of CTX-M allele or TEM type.Were they identical or were different allele types of the resistance genes found?A more detailed discussion on that point should be included.

Review 1:
Notes: Introduction lines 82-86.All the different classes of beta-lactamases are listed by their acronyms, eg.OXA, SHV etc.But what is not listed is what they stand for, ie.Oxacillinase (OXA).What is not listed is the class B type, metallo beta-lactamases in the introduction.The authors should note that beta-lactamases have four classes, and OXA is class D. This needs to be made clearer.
Response: Thank you for pointing this out.We have thoroughly reviewed the addressing of beta-lactamases in the Introduction section, referencing Bajpai T, Pandey M, Varma M, Bhatambare GS. "Prevalence of TEM, SHV, and CTX-M Beta-Lactamase genes in the urinary isolates of a tertiary care hospital," published in Avicenna J Med in 2017; 7:12-16, and Sharma J, Sharma M, Ray P. "Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India," found in the Indian J Med Res in 2010; 132:332-36.We have defined each abbreviation of beta-lactamase in line 84-89 of the revised manuscript.
Results line 236-7.The sentence reads as though all isolates contained all three bands, but I'm sure the authors mean that all 199 isolates were positive for at least one band, not all three.Otherwise, the rest of the analysis does not make sense.
Response: We have edited and highlighted the text in lines 242-244 of the revised manuscript.
In the materials and methods section, it is mentioned that "ESBL isolates with a similarity coefficient {greater than or equal to} 85% were considered the same genotype (20)."How many of the 199 isolates were considered the same genotype?
Response: A dendrogram was constructed using the unweighted pair group method with arithmetic mean.This grouped the isolates into clusters with greater than 85% similarity, with the scale indicating the percentage of similarity.We have incorporated these critical d etails into the manuscript.184(92.5%) were considered the same genotype A, 3 (1.5%)were considered B type, and unique type were 12 (6.0%).We have edited Table 2 and other relevant details.
Discussion line 263-265: Place the reference next to the reported percentage, rather than at the end of the sentence.
Response: Thank you.As suggested, we have placed the reference next to the reported percentage in line 272 and highlighted the sentence in the Discussion section.
It's good that the authors acknowledge the limitations of the study.They should also note that there's a strong chance that many of the isolates are clonal, hence the fact that the ERIC-PCR result shows that nearly all the isolates are from group A (and 100% of the K. pneumoniae ones were).The strong possibility that many of the isolates are clonal needs to be mentioned here (lines 302-316).
Response: Thank you for your insight and comment.Accordingly, we have expanded the necessary details in the revised manuscript in lines 326-328.

Figures:
The labelling  Response: We have removed the blurred text, and regarding the concerns about the 300 dpi resolution of the figures, our images were generated using the BioNumerics 7.0 software at the maximum resolution available in dpi.Additionally, we have included a code of ESBL isolates (Figure 2) and all data were submitted to the NCBI (BioProject No. PRJNA984445.
We appreciate your attention to detail.The sample of ERIC-PCR profiles presented in below were analyzed with Bionumerics software, employing Pearson's correlation coefficient to evaluate the similarity among band patterns.Subsequently, a dendrogram was constructed using the unweighted pair group method with arithmetic mean.This grouped the isolates into clusters with greater than 85% similarity, with the scale indicating the percentage of similarity.We have incorporated these critical details into the manuscript.
We have a representative gel image for ERIC-PCR from the Bio-Rad Gel Doc XR Imaging System for capturing agarose gels.Lane M represents molecular marker (100bp), lane 1-16 represents samples showing characteristic ERIC banding pattern.
For tables 4 and 5, state which test you are using to get a p-value (student t-test, odds ratio?) Response: We have provided independent sample t-tests to compare the values of continuous variables between groups.Statistical significance was measured using two -tailed tests, and significance was set at P < 0.05 in line no.249-250 on page no. 6.
Overall, the paper reads well and the authors do analyse the data they have presented in a clear and concise manner.The figures need to be improved for publication quality (see above).It is good to know that most of the isolates are carbapenem sensitive, probably owing to the lack of bla PER alleles identified.
Response: We appreciate your insightful point.We have edited the details, as suggested.
One other point, the authors state that they sequenced the alleles.But I did not read any analysis on whether all isolates had the same type of CTX-M allele or TEM type.Were they identical or were different allele types of resistance genes found?A more detailed discussion on that point should be included.
Response: CTX-M and TEM alleles have identical allele types.bla CTX-M was detected in 1 (1.3%)K. pneumoniae and 61 (50.8%)E. coli isolates.Table 4 shows that bla TEM was detected in 57 : it is not clear what the p-values correspond to/measure Introduction lines 82-86.All the different classes of beta-lactamases are listed by their acronyms, eg.OXA, SHV etc.But what is not listed is what they stand for, ie.Oxacillinase (OXA).What is not listed is the class B type, metallo beta-lactamases in the introduction.The authors should note that beta-lactamases have four classes, and OXA is class D. This needs to be made clearer.
for figure one needs to be improved.State in the legend which lanes have which samples and remove the written text from the gel as it is very confusing.Also state what size each band should be in the figure legend.

Figure 2 :
Figure2: It is very hard to read figure 2. It looks like there is text at the end of each line on the dendogram, but it is impossible to read it.Either remove the text or find a way to identify the isolates in an easier way (ie.Different colours for E. coli or K. pneumoniae).Also, could a gel of the ERIC-PCR be included in the figure?
for figure one needs to be improved.State in the legend which lanes have which samples and remove the written text from the gel as it is very confusing.Also state what size each band should be in the figure legend.Response: Thank you for pointing this out.We have edited Figure2and included the necessary details.

Figure 2 :
Figure 2: It is very hard to read figure 2. It looks like there is text at the end of each line on the dendrogram, but it is impossible to read it.Either remove the text or find a way to identify the isolates