Investigating Antimicrobial Resistance and ESBL Producing Gene in Klebsiella Isolates among Neonates and Adolescents in Southern Bangladesh

Background Multidrug-resistant (MDR) clones of Klebsiella pneumoniae (Kpn) have been increasingly documented in community-acquired and nosocomial infections all around the globe. Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamase enzymes derived from SHV genes by mutations. This research work aimed to investigate and analyze the widespread prevalence of Kpn antibiotic resistance in different areas of the southern part of Bangladesh. Methods This particular study was executed and implemented by using 501 clinical samples or isolates from two different hospitals in Chattogram. The disk diffusion method was used to detect Kpn's sensitivity to 16 antibiotics in a drug susceptibility test. By using the PCR technique, the widespread prevalence of antibiotic-resistant gene blaSHV-11 was studied. Sequencing along with phylogenetic analysis was utilized to verify isolates with the blaSHV-11 gene. Results Almost all of the Kpn isolates were spotted to be antibiotic-resistant. These Kpn isolates were resistant to β-lactams, aminoglycosides, and quinolones at high levels. The spatial analysis displayed that infections involving Kpn were more common in the urban areas (70%) than in the rural areas (30%). Neonates had substantially higher levels (p < 0.001) of resistance to multidrug than other age groups. Cefepime was identified as the most frequent antibiotic-resistant to all age groups (56.68%). The highest numbers of resistant isolates (36.92%) were found in urine samples. The ESBL gene blaSHV-11 was found in 38% isolates. Conclusion The significant frequency of MDR Kpn harboring β-lactamases and AMR genes strongly suggests the requirement to develop effective antimicrobial resistance control and prevention measures in Bangladesh.


Introduction
Antimicrobial resistance (AMR) is a global burden affecting multiple sectors [1,2]. Antibiotic resistance will cost the global economy $100 trillion by 2050, with a death rate of nearly 10 million people each year, surpassing cancer and heart disease [3,4]. monitoring, implementing current policies and initiatives for the issue of antibiotic resistance remains an obstacle. Bacterial genes that resist antibiotic action, known as resistance genes or "resistomes," contribute to bacterial resistance against antibiotics on a global scale. Resistomes are highly widespread, tenacious, and can easily spread from a single type of bacterial strain to another belonging to a completely different genus through mechanisms such as gene transfer through horizontal techniques. As a result, antimicrobial resistance poses a worldwide concern [6,7]. Kpn has been reported to be significantly resistant to the four main kinds of antibiotics, resulting in both MDR (multiple drug resistance) and XDR (extreme drug resistance) type Kpn development, as per the European Antimicrobial Resistance Surveillance Network [8].
e SHV-11 (sulfhydryl reagent variable) gene, which belongs to the ESBL genes' SHV-type variants, was demonstrated to have lactamase activity against beta-lactams such as penicillin and well-known third-generation antibiotic cephalosporins [11]. Kpn strains possessing the chromosomal SHV-11 β-lactamase gene are more likely than those carrying the chromosomal SHV-1 β-lactamase gene to generate the plasmid-mediated SHV-12 extendedspectrum lactamase [12]. e β-lactamase generating ESBL genes were detected in most of the multidrug-resistant isolates [13].
As a result of the fast appearance and spread of MDR superbugs, resistance to antibiotics in microorganisms has now become an impending global issue that can cause major damage. e objective of this research study was to determine the widespread presence and genetic changes of the gene that produces ESBL and the respective SHV-11 variant form in newborns and children (ages 0-18 years) infected with Kpn in Bangladesh. Understanding antibiotic resistance in commonly found microorganisms on a molecular level can provide opportunities for early management and implementation of proposed strategies before the consequences become too catastrophic.

Kpn Strains and Phenotypic
Tests. By following the CLSI (Clinical Laboratory Standard Institute) standards, culture characteristics, regular standard biochemical assays, and Gram stain methods were used to evaluate the Kpn-containing isolates. First, chocolate agar, Mac-Conkey agar medium, and blood agar were used to examine the shape as well as the colony of bacterial species. en, gram stain methods were used to differentiate bacterial groups. After confirmation analysis of Klebsiella pneumonia (Kpn) by subculture method, a variety of standard biochemical tests were conducted (Voges Proskauer, indole, urease tests, TSI [triple sugar iron], motility, citrate, and methyl red) [14][15][16].

Molecular
Confirmation of blaSHV-11. For molecular identification of blaSHV-11, Kpn isolates' genomic DNA with the highest incidence of resistance to antibiotics was isolated by using the usual boiling technique [19,20]. In a NanoDrop ( ermo Scientific) Spectrophotometer 2000, the concentration and purity of the isolated DNAs were evaluated at 260/280 absorbance. Polymerase chain reactions (PCR) were used to identify the blaSHV-11 gene from confirmed MDR Kpn isolate by using the primers listed below: Forward_5′-ATGCGTTATATTCGCCTGTGTATT-3′; Reverse_5′-GCGTTGCCAGTGCTCGATCAGCGC-3′ [21,22] (annealing temperature 51.2°C) [21]. An entire amount of 25 μL aliquot, where 12.5 μL was a green master mix, 1 μL Reverse and Forward primer each, 1 μL Template DNA, and the rest was nuclease-free water, was set for PCR at 51.2°C by using a thermal cycler (Nyx technology).

Gel Electrophoresis.
One gram agarose was dissolved into 100 ml of 1X (TAE) tris-acetate-ethylene-diaminetetra-acetate for preparing a 1% (w/v) agarose gel. e agarose solution was heated in a microwave for around 3 minutes and then kept to be cooled to approximately 50°C. e molted transparent gel was poured into a casting tray having a comb set up and allowed to be semisolidified. e comb was removed after 20 minutes, and the tray holding the gel without the comb was kept in a gel electrophoresis chamber filled with TAE buffer, with 5 μL of each amplified product inserted into a separate well of the agarose-made gel. A 5 μL extended quick ladder and nuclease-free (NF) water as negative control were also added. For around 50 minutes, an electric current of 80 V and 400 mA (milli Ampere) was passed through the 1% gel electrophoresis chamber, after which the gel was submerged in an ethidium bromide-containing box for 30 minutes before being visualized and photographed by using UV light in the gel documentation chamber.

Sequence Analysis.
e purified PCR products of bla

Phylogenetic Analysis and Evolutionary Relationships of
Taxa. By observing the tree, it became obvious that two of our sequences (MN551177.1 and MN551175.1) had significant variations (Figure 4). For that reason, they were observed to be clustered with each other. ough the other two Kpn sequences of this study were also observed to be clustered together, there was a minor variation between them. e source of each gene was enclosed in Supplementary Table 1, including accession ID, isolation source, year, and host. is study thus illustrated changes among all the sequences, but it needs further study with more sequences from Bangladesh to understand clustering in depth.

Discussion
In recent times, Kpn has emerged and spread as an opportunistic, drug-resistant pathogen, creating grave clinical concern [22]. Its prevalence has expanded to a variety of niches, from mucosal layers of animals and humans to water from sewerage channels, soil, and even vegetation [22,23]. Similarities in biochemical properties, virulence, and pathogenicity were noticed in environmental origin and clinical origin-based Kpn strains [22,23]. In terms of antibiotic resistance, clinical Kpn strains have been determined to be more resistant to several types of antibiotics than those found in the environment [24].
First of all, a higher number of resistant strains were recovered from the specimens that were taken from urban patients than from rural ones. Secondly, since the number of pharmacies in the urban areas is higher than that in the rural ones, the random use of antibiotics is significantly rising there. e pattern is also extending into the aquatic and food chains. As a result, secondary infections brought on by resistant types of bacterial strains are also occurring more frequently. Because of data gaps and subpar medical care, the genuine reality of rural areas is not revealed here. Kpn infection rates were noticeable in both females and males in this particular research work, especially with multidrug-resistant strains of Kpn. Kpn isolates are often the most encountered pathogen responsible for acute pyelonephritis during childhood [25]. However, Kpn causes pyelonephritis in females and upper UTI in males >20 years old [26].
Among the various age groups considered for the research work, toddlers (12 months-36 months) exhibited the highest MDR (81.16%), followed by adolescents (12-18 years) (73.33%). MDR was spotted in all other age groups, indicating a significant prevalence of resistance to antimicrobial drugs in children and neonates in general. ese data appear to support the general consensus that resistance to antimicrobial drugs is on the rise both nationally and globally [27,28]. e widespread usage of antimicrobial drugs, along with an undeveloped immune system in children and neonates, creating selective pressure, appears to be the major cause of MDR bacterial strains comparable to Kpn infections in Bangladesh. Poor hygiene practice, sanitation, and subpar infection control among children and neonates in healthcare settings are some of Bangladesh's most likely reasons for antibiotic resistance [29].
Kpn infection rates among neonates and children are noticeably high, as seen in this current study. In hospitals, children with malnourished conditions have higher antibiotic resistance for infections such as Kpn infection due to dehydration, acute wasting, etc. ese health conditions among children, neonates, and adolescents alter gut functions and damage mucosal levels which makes it easy for Kpn to translocate to other places, for example, the kidneys from the gut [30]. In Bangladesh, hospitals experience high numbers of neonates, child patients with malnutrition diseases, and infections of the kidneys or the urinary tract, which make it easier for Kpn along with other bacteria, for example, E. coli, to spread internally and cause community infections [31].  In this work, higher infection rates and resistance rates were observed among patients in hospitals (60.5%) which may indicate hospital-acquired resistance to antimicrobial drugs among patients (Table 1). Prior research work found that Kpn samples recovered from clinical equipment, hands/ clothes of medical staff, and clinical samples from the hospital, such as the blood and urine, were more resistant than the general rate [32]. e high mutation rate of Kpn along with its fast spread among communities over time could be a reason for hospitalized patients having bacterial strains with heightened levels of MDR. Studies also support that the colonization rate of Kpn was revealed to have increased in patients in direct proportion to the duration of their hospital stay. Rates of colonization in hospital-staying patients were four times higher in carriers in comparison to noncarriers of bacterial infection [23].
Kpn spreads throughout the body from the gut in both adults and children. It is found in sputum and urine samples because these samples come from areas where frequent bacterial turnover occurs, such as kidney infections or UTIs [33]. Mechanisms used by transposable elements and plasmids of Kpn strains help it to spread and cause infections such as nosocomial pneumonia and UTI [34].
In our research work, MDR was exhibited in the majority (70%) of the Kpn sample isolates. We also identified that these isolated samples were not susceptible or rather greatly resistant to common antibiotics used either singularly or in association with other drugs to treat Kpn infections, such as β-lactam antibiotics-2nd and 3rd generation of cephalosporin, aminoglycosides, macrolides, and quinolones (Figure 2). Causes and risk elements that support this pattern of resistance to antimicrobial drugs include gene transfer through a horizontal method, incomplete cycles of medicine intake, taking nonprescribed antibiotics, limited diagnostic facilities, limited monitoring and surveillance systems for patients, immunocompromisation, rapid community infections, and antibiotic overuse in organic produce [17,[35][36][37].
Kpn's spatial distribution among neonates and young adults in our work suggests that MDR is prevalent in this population in both rural and urban regions (Figure 3). MDR infections account for around 70% of all Kpn infections. When compared to equivalent figures in a developed country, this is extremely high. According to one particular recent research from Public Health England, around 21% of patients suffering from bloodstream infections are diagnosed with resistant key pathogens [38]. During 2015 and 2019, there was a 32.5% elevation in resistant infections, as reported by the same report. However, since our study depicts cross-sectional data for the Kpn mediated MDR burden in Chattogram only, further conduction of a systematic study on a larger sample and location span is necessary to appraise the actual burden of resistant pathogens and find out the cause to further develop a sustainable mitigation plan to tackle the MDR related complications. In this research work, several clinical isolates (38%) were SHV-11 positive, showing a highly resistant pattern to the bulk of the clinically used antibiotics. Neonates and infants were most prone to infection. Previously, this gene was reported in water samples, drain water, and bed trails from the environment [39]. In a previous study, in MDR, Kpn isolates SHV-12 were discovered from surface water [40]. Prolonged hospital stay, ICU, and invasive positive ESBL isolate input through central venous, urinary, and endotracheal catheters are all risk factors for ESBL-containing isolate dissemination [41].
From the evolutionary tree, it is apparent that MN551177 is one of the most ancient genes of Kpn species (Figure 4). It shared a common ancestry with a strain of accession number HBL89233.1. Due to the high sequence conservancy of the recently discovered gene mutation of SHV-11 in Chittagong, MN551175, MN551177, and MNMN437452 showed a cluster in the tree. Yet, the first two of these clusters are of very common ancestor on account of their own sequence in two directions, AVO16696.1.
Bangladesh is particularly vulnerable to the ramifications of AMR and the dissemination of MDR pathogens due to a lack of knowledge about the proper use and dose of various antibiotics. is included selling off and taking prescription antibiotics without a physician's advice, antimicrobial drug usage in cattle feed and not completing a full cycle of antibiotic dosage [42][43][44]. roughout the country, antimicrobial drugs are readily accessible and can usually be taken without a proper prescription, resulting in a spiral in their irrational use across industries, resulting in pollution and uncontrollable antimicrobial resistance dissemination [40,42,45,46].
Although the results obtained from this study are substantial, it should be taken into account that the study was conducted with a limited number of samples and was solely based on patients from two hospitals in southern Bangladesh who were unable to exhibit the complete picture of the whole country. Despite these limitations, this study will serve as a benchmark for further work on antibiotic resistance among neonates and children in Bangladesh.

Conclusion
In this study, significant prevalence of Kpn isolates resistant to antimicrobial drugs was observed among neonates and children. Moreover, high occurrence of Kpn strains with a considerable distribution of antibiotic-resistant genes was reported. Existence of the ESBL-producing gene, and an elevated persistence of antimicrobial drug resistance to various antibiotic classes, raise a serious public and community health concern regarding the worsening condition of hospital infections. To be able to fully assess the epidemiological implications of Kpn strains and their effects, it is high time to consider conducting molecular studies on larger study samples. Identifying the prevalence of virulence factors in various strains of Kpn will help to design new therapeutic option and implement more efficient policies for preventing a future pandemic related to superbugs.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
AM, AA, WA, and MMH: conceptualized the study. AA, NS, MSH, SN, and MMH the methodology, investigation, and validation. AM, AA, MSH, and SN wrote original draft preparation. AM, AA, NS, WA, and MMH: wrote, reviewed, and edited the manuscript. NS, MMH, and WA: resources. AM: acquired of the financial support for the project leading to this publication.  Figure 4: Molecular Phylogenetic analysis of SHV-11 protein. e evolutionary history was inferred by using the maximum likelihood method based on the HKY model. Here, the tree was built with the bootstrap value (100). e tree was drawn to scale, with branch lengths measured in the number of substitutions per site.