Molecular Epidemiological Surveillance of CTX-M-15-producing Klebsiella pneumoniae from the patients of a teaching hospital in Sindh, Pakistan.

Background The presence of Extended-spectrum β-lactamase positive bacteria in hospital setting is an aggravating influential factor for hospitalized patients, and its consequences may be hazardous. Therefore, there is a need for rapid detection methods for newly emerging drug-resistant bacteria. This study was aimed at the molecular characterization of Extended-spectrum β-lactamase -positive Klebsiella pneumoniae isolates recovered from the patients of a teaching hospital in Sindh, Pakistan. Methods A total of 513 K. pneumoniae isolates were obtained from various clinical samples during June 2019 to May 2020. The collected isolates were investigated for antimicrobial susceptibility (antibiogram), and PCR and DNA sequencing were performed to analyse the ESBL genes. Results Among the 513 isolates, as many as 359 (69.9%) were Extended-spectrum β-lactamase producers and 87.5% were multi-drug resistant, while none had resistance to imipenem. PCR scored 3% blaTEM, 3% blaSHV, and 60% blaCTX-M-15 genes for the tested isolates. Conclusion The study showed that CTX-M-15 was the major prevalent Extended-spectrum β-lactamase type among the isolates. Additionally, all the isolates were susceptible to carbapenems. Screening and detection of Extended-spectrum β-lactamase tests are necessary among all isolates from the enterobacteriaceae family in routine microbiology laboratory to prevent associated nosocomial infections. A larger study is essential to understand molecular epidemiology of Extended-spectrum β-lactamase producing organisms to minimize morbidities due to these multidrug resistant organisms.


Introduction
The emergence of Extended-spectrum β-lactamase (ESBLs) producing organisms has imposed a great threat on majority of the antibiotic classes, particularly cephalosporins. 1 The situation worsens when the patient infected with ESBLproducing organism is administered an antibiotic to which the organism is resistant. 1 Clinical practitioners globally have been facing problems related to plasmid mediated ESBL producing Klebsiella pneumoniae (K. pneumoinae). These plasmids contain genes encoding resistance to antibiotics such as aminoglycosides, sulfonamides, tetracycline, chloramphenicol, and quinolones. 2 Cephalosporins are β-lactam antibiotics that are widely used in clinical practice and in the treatment of bacterial infections. 3 In addition, K. pneumoniae is reported in nosocomial infections and community acquired infections. 4 A study conducted by Maltezou et al. 5 revealed that the incidence rate of ESBL producing K. pneumoniae infection was 16.71% for Northern Europe, 24.41% for Southern Europe, 58.71% for Eastern Europe, 28.2% for the Asia Pacific region, 51.9% for South America, and 12.3% for North America. 5 CTX-M-type β-lactamase, has become more predominant than conventional SHV and TEM-type ESBL, which have covered an extensive range of clinically significant bacteria and over a wide environmental area. 6 Moreover, strains that yield ESBL often reveal resistance to antibiotics belonging to other classes (i.e. aminoglycosides, quinolones, and sulfonamides); making its management more complicated. 7 Consequently, this class of bacteria makes a need to ensure the reporting of ESBL producers in clinical isolates and detection of newly emerging drug resistant isolates. The changing trends of drug resistant ESBL-producing K. pneumoniae need time to watch for management, confinement and isolation of patients in hospitals before discharge. It is also necessary to monitor the prevalence and types of ESBLs and define the appropriate therapeutic options accordingly. Hence, the purpose of the current study was to observe the current trends of ESBL producing K. pneumoniae drug resistant patterns and to identify the occurrence of ESBL genes in Sindh region of Pakistan.

Sample collection, inclusion and exclusion criteria
A total of 1458 non-duplicate clinical samples were collected between June 2019 and May 2020 from outpatient and inpatient wards of People's University of Medical and Health Sciences, Nawabshah, Pakistan [tertiary care government hospital with 1000 beds] through its diagnostics and research laboratory. Patients referred by their primary physicians for screening tests (such as blood complete count and erythrocyte sedimentation rate (ESR) were recruited in the study. The clinical samples including blood, sputum, urine, cerebrospinal fluid, tracheal secretions, and pus samples were collected by standard techniques ( Figure 1A). The clinical samples, which yielded the K. pneumoniae were selected for further assessment, while others were excluded. The demographic data was retrieved from clinical survey that included age, gender, specimen type, town, and susceptibility patterns ( Table 2). ; genotypic distribution showed blaCTX-M-15 to be highly prevalent (B); age group wise analysis showed elderly group of 50 years old and above showed high incidences (C); ESBL positivitiy to be 70% among all isolates tested (D), with their antibiotic susceptibility profile (E).

Isolation and identification
The samples were not specifically collected for this research, they were used after the completion of routine laboratory investigations upon the permission of the concerned laboratory's authority. The isolation of bacteria was carried out by conventional bacteriological techniques and identification was confirmed by API 20E identification kit (BioMerieux, France). 8 As many as 513 K. pneumoniae isolates from various clinical samples and sites ( Figure 1A) were obtained and included in the present study.
ESBL screening and confirmation by phenotypic methods Double disk diffusion methods were used to detect ESBL using four antibiotic disks: cefotaxime 30 μg, cefotaxime with clavulanic acid 10 μg, ceftazidime 30 μg, and ceftazidime with clavulanic acid 10 μg. A more than 5 mm increase in zone of inhibition width for whichever antimicrobial agent tested in combination with clavulanic acid against its zone of inhibition when tested alone was labelled as ESBL positive. 9 The inoculum and incubation conditions were the same as recommended for standard disk diffusion method. K. pneumoniae ATCC 700603 (American type culture collection, ATCC, Manassas, USA) was used as quality control strain.

Detection of ESBL genotypes by PCR amplification
Bacterial genomic DNA was seperated by boiling the young bacterial colony in 50μl distilled water in a hot bath set at 98°C. This DNA template was used for all PCR reactions. Amplification of blaTEM, blaSHV and blaCTX-M-15 ESBL genes was done on GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The primers used are given in Table 1. The PCR yields were run on 1% agarose gels and observed in UV light. Purified PCR products of CTX-M genes were sequenced on an Applied Biosystems 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA).
Reaction mixture: A 30 μl total reaction mixture consisted of the following: 2 μl of DNA template; 1.5 μl of 10 μM forward and reverse primer; 1.5 μl of Sigma brand Red Taq; 20.2 μl of pure water; 3 μl of PCR buffer with MgCl 2 ; and 0.3 μl of 10 mM dNTPs.

Data analysis
The data were analyzed by IBM SPSS Statistics for Windows, Version 21.0, Inc. USA Statistical Package of Social Sciences (SPSS) version 21.0. Continuous and categorical variables were analyzed by t-test and Chi-square testing, respectively (these tests can also be carried out on the open source software R). Variance of resistance levels of various antimicrobials in ESBL producing isolates versus non-ESBL producing isolates were calculated by the Fisher exact test. The data was analyzed at 95% Confidence interval (p ≤ 0.05 indicates a significant difference).

Results
During the study period, a total of 513 (35.18%) K. pneumoniae were isolated. From those 513 positive cultures, 411 (80.1%) belonged to males and 102 (19.8%) to females (p = 0.0001). The demographic characteristics of patients including age, gender, rural or urban, and clinical interventions are summarized in Table 2. The results show that the majority of patients were in the older age group (50 and above) and lived in urban areas Figure 1B. The categories of patient's ages have been shown in Figure 1C. The mean age of the total study subjects was 48.5 AE 8.9 years.
Out of 513 isolates 359 (69.9%) were ESBL producing K. pneumoniae ( Figure 1D). The incidences of ESBL was higher in males as compared to females. The frequency of ESBL producing K. pneumoniae from different samples (n = 359) has been shown in Table 3 and Figure 1A. The means AE SD age of patients with ESBL positive and negative K. pneumoniae was 47 AE 17.5 years and 47.5 AE 12.8 years, respectively (p = 0.093).
The results show that 64% of K. pneumoniae in our sample were resistant to Amoxicillin and Cefixime. In addition, an important drop of 30-40% was perceived in the susceptibility for all Cephalosporins. However, K. pneumoniae presented a dissimilar sensitivity rate to Chloramphenicol, Ofloxacin, Gentamicin and Amikacin with 94%, 98%, 97%, and 97%, respectively and depicted in Table 4.
Regarding the PCR and DNA sequencing of ESBL genotypes, it was found that all of the ESBL-positive K. pneumoniae isolates had one or more ESBL genes that were tested in the present study and presented in Table 5. Overall, 83.56% (300/359) of K. pneumoniae isolates were positive for one or more ESBL genes. The PCR assay and DNA sequencing results indicated the following frequencies of ESBL genotypes: 85% blaCTX-M-15 gene, 26% blaSHV gene, and 28% blaTEM gene.

Discussion
ESBL's are the major causes of β-lactam antibiotic resistance, particularly in K. pneumoniae, which is amongst the most common gram-negative bacteria belonging to Enterobacteriaceae families. 10 A study from Ziauddin University Karachi, Pakistan has showed that ESBL producing K. pneumoniae frequency is 84.16% amongst reported cases. 11 Similarly, Saleem et al. (Aga Khan University, Karachi, Pakistan) have reported a frequency of 80% of ESBL producing K. pneumoniae. 12 These findings were higher as compared to the results presented in the current study, which showed that the frequency of ESBL producing K. pneumonia is 69.6% with a higher incidence in male than in female patients. In this repect, the frequency of EBSL producing K. pneumoniae varies from one institution/hospital to another. Several factors could govern this variation such as; the use of antibiotics, drug dose, infection control measures, treating physicians, and the duration of drug therapy -all of these elements may cause a difference in results. The frequency of ESBL producing K. pneumoniae in previous research conducted at Aga Khan University Karachi, Pakistan was 30.1% and 47.8%, which is low compared to the present study, as well as, previous studies conducted by Mansouri et al. 10 and Ejaz et al. 13 A Pakistani study found a frequency of ESBLs producing K. pneumoniae of 70%, 14 which is in agreement with our study.
Carbapenems are often the final influential therapy of choice to treat infections resulting from MDR Enterobacteriaceae. Based on our study and other studies, 100% sensitivity was seen with Imipenem, which have been found to be the most effective antibiotics on the isolates that produce ESBLs. This is an important result of the present study because many infections can be treated with Carbapenems. 12,15 blaCTXÀM is a predominant genotype in this area of the subcontinent. A further study from Pakistan has reported that 72% of ESBL producing strains had the blaCTXMÀ15 gene, which was higher than the prevalence of blaCTX-M gene  reported in the present study. 16 Limited studies from different regions have also shown a higher prevalence of the blaCTX-M genotype amongst strains including 84.7% (Chile), whereas a very high prevalence of 98.8% was reported in China and, significantly, a low prevalence of 13.6% in Tanzania. [17][18][19] In conclusion, the findings of the current study emphasizes the urgent need for screening and surveillance of ESBL producing K. pneumoniae in routine microbiology laboratories for early detection, prompt intervention, and successful clinical management before exacerbation of the infection magnitude. In this regard, the current findings may be a valuable contribution to the medical literature providing physicians with an updated prevelance status of ESBL producing K. pneumoniae. Larger studies need to be done in various geographical regions of the country to better define the molecular epidemiology of ESBL-producing K. pneumoniae and its clinical implications. 20 Finally, the limited sample size along with the limited time frame are two common limitations, which could affect the outcomes of the study.

Mubashir Zafar
Aga Khan University, Karachi, Pakistan The article is good but it needs major revision.
First, the title of the article should be revised as Epidemiological Surveillance of CTX-M-15producing Klebsiella pneumoniae among patients in teaching hospital (add city and country name).

Major revision Abstract
The objective is different from the title of the study. ○ Don't use the abbreviation in the abstract.

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The method in the abstract is incomplete.

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The result and conclusion are also incomplete information in the abstract.

Minor revision
The article would benefit from being revised for formatting and use of language.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? No
Competing Interests: No competing interests were disclosed. There is no mention about ESBL positive K. pneumoniae in the Figure 1A description/legend, whereas the same has been described in the running text. unaddressed Clinical relevance of demographic characteristics with the results obtained in this study is not described clearly by the authors in discussion section. This needs to be elaborated." unaddressed As per the study it's not only Carbapenems but also quinolones (Ciprofloxacin) also that showed their effectiveness with 100% sensitivity. Authors has missed to point it out and describe about it in the running text. unaddressed Relevant references needs to be quoted for conventional bacteriological techniques in methods unaddressed As per the Fig 1E, the authors have tested different classes of antibiotics, here what was the rationale behind choosing these specific antibiotics? The results illustrates that the more percent resistance was mainly observed in the antibiotics belongs to class of cephalosporins, is there any clinical relevance for this kind of observation, this needs to be discussed in the discussion section with relevant references if available. unaddressed Authors mentioned in the method that the genomic DNA was separated upon boiling the bacterial colony at 98 degrees Celsius and the same DNA was used for PCR amplification of ESBL genes. The ESBL genes are mainly encoded by plasmid DNA. Here, in this study the authors used which DNA (bacterial genomic DNA or plasmid DNA) for PCR amplification to understand the genetic diversity of ESBL genes Needs to be addressed The highest percent of ESBL was found in blood specimens, is there any scientific reason for this? If yes, it needs to be described in the discussion part. Unaddressed Authors mentioned in the method that the genomic DNA was separated upon boiling the bacterial colony at 98 degrees Celsius and the same DNA was used for PCR amplification of ESBL genes. The ESBL genes are mainly encoded by plasmid DNA. Here, in this study the authors used which DNA (bacterial genomic DNA or plasmid DNA) for PCR amplification to understand the genetic diversity of ESBL genes? Unaddressed

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility?

Are the conclusions drawn adequately supported by the results? No
No Please add company and country for SPSS software.

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The sequenced products of the isolates need to be deposited in a database (i.e. Genbank) and accession numbers need to be given.

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Provide the accession numbers of the sequenced products in the article.

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In the title of the article the authors have mentioned that isolates collected from inpatients and outpatients of a teaching hospital, but in the main text is no mention of them and the number of isolates in each group is not mentioned? Please explain.
○ Finally, I recommend merging both Results and Discussion sections for better clarity and impact, and move to Rapid Communication submission.
We thank the reviewer for taking time and reviewing our article. We have revised our

Finally, I recommend merging both Results and Discussion sections for better clarity and impact and move to Rapid Communication submission.
section need to be removed.
Author mentioned Kirby bar method, it needs to be mentioned as Kirby Bar Disk diffusion method.

5.
Under methods section in the paragraph on ESBL screening and confirmation by phenotypic methods, in the last but one line the standard disc diffusion where "method" is missing needs to be written.

6.
There is no mention about ESBL positive K. pneumoniae in the Figure 1A description/legend, whereas the same has been described in the running text.
7. Figure 1B is not described in the running text. Also in figure there is confusion with the numbering corresponds to the genes, for instance in TEM, SHV and CTX-M there should be three numbers (one number for each gene) but only two numbers are given along with the percentage. Please check and confirm.

8.
Clinical relevance of demographic characteristics with the results obtained in this study is not described clearly by the authors in discussion section. This needs to be elaborated.

9.
As per the study it's not only Carbapenems but also quinolones (Ciprofloxacin) also that showed their effectiveness with 100% sensitivity. Authors has missed to point it out and describe about it in the running text. 10.
The highest percent of ESBL was found in blood specimens, is there any scientific reason for this? If yes, it needs to be described in the discussion part.

11.
What is the denominator for 411 males and 102 females clinical specimens from whom K. pneumoniae were isolated? If the sample sizes for male and female donors were different then before coming to the conclusion saying that in males it was higher than in females it needs to be proved statistically. Authors needs to clarify this.

12.
As per the Fig 1E,  There is no mention about ESBL positive K. pneumoniae in the Figure 1A description/legend, whereas the same has been described in the running text.

Author's Response:
The aim and objectives of the study were to perform molecular epidemiology of the isolates regardless of their clinical sources and clinical presentations. This was beyond the scope of clinical correlations. However, existing data were being used for further study and analysis where additional publication is being tried.

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As per the study it's not only Carbapenems but also quinolones (Ciprofloxacin) also that showed their effectiveness with 100% sensitivity. Authors has missed to point it out and describe about it in the running text.