Prevalence and molecular characterization of antibiotic resistance and associated genes in Klebsiella pneumoniae isolates: A clinical observational study in different hospitals in Chattogram, Bangladesh

Afroza Akter Tanni; Md Mahbub Hasan; Nahid Sultana; Wazir Ahmed; Adnan Mannan

doi:10.1371/journal.pone.0257419

. 2021 Sep 10;16(9):e0257419. doi: 10.1371/journal.pone.0257419

Prevalence and molecular characterization of antibiotic resistance and associated genes in Klebsiella pneumoniae isolates: A clinical observational study in different hospitals in Chattogram, Bangladesh

Afroza Akter Tanni ¹, Md Mahbub Hasan ¹, Nahid Sultana ², Wazir Ahmed ³, Adnan Mannan ^1,^*

Editor: Monica Cartelle Gestal⁴

PMCID: PMC8432802 PMID: 34506611

Abstract

Objective

This study was performed to investigate the prevalence of multidrug resistance and molecular characterization of Klebsiella pneumoniae (KPN) from clinical isolates in the southern region of Bangladesh. Additional analysis of the prevalence of bla_NDM-1, bla_SHV-11, uge genes of KPN was also carried out among these clinical isolates.

Method

The study was carried out using 1000 clinical isolates collected from two different hospitals of Chattogram. A drug susceptibility test was performed by the disk diffusion method to detect KPN’s response to 16 antibiotics. The presence of antibiotic-resistant and (or) virulent genes bla_NDM-1, bla_SHV-11, uge were investigated using the PCR technique. Isolates having bla_NDM-1, bla_SHV-11, uge gene were further validated by sequencing followed by phylogenetic analysis. Phylogenetic relationships among these isolates were determined by Clustal omega and MEGA7.

Result

A total of 79%, 77%, 74.9%, 71%, 66% and 65% isolates exhibited resistance against cefuroxime, cefixime, cefotaxime, ceftazidime, cefepime and ceftriaxone respectively. The frequency of resistance to other antibiotics varied from 26.5% to 61.8%. PCR analysis showed that 64% of strains harbored bla_NDM-1 gene, and 38% strains harbored bla_SHV-11 gene. Moreover, 47% of samples were carrying uge gene, and 19% of samples carried bla_NDM-1, bla_SHV-11, uge genes together.

Conclusion

In this study, we’ve analysed the pattern of expression as well as prevalence of bla_NDM-1, bla_SHV-11, and uge genes in Klebsiella isolates. Upon molecular and statistical analysis, we found a high prevalence of multi-drug resistance KPN strains in the isolates. The Klebsiella isolates were confirmed to harbor multiple ESBL genes and 64% of the isolates were found to be producing NDM-1. As multidrug resistance is an alarming issue, continuous surveillance and routine clinical detection of resistant bacteria and plasmids are necessary to prevent catastrophic public health incidents.

1 Introduction

Since the discovery and commercialization of antibiotic drugs, preventing and treating pathogen mediated and driven diseases have become comparatively more accessible. However, this ceaseless use of antibiotic drugs has been subject to abuse as well through irregular administration and overdose in veterinary, livestock breeding, and farming [1–3], which has caused multiple pathogens to develop resistivity against antibiotics. Over the years antimicrobial resistance (AMR) has become one of the major concerns of global health issues [4].

Given the gravity of the AMR issue, data relevant to AMR is being collected and analyzed constantly through clinical studies in multiple populations. Yet, according to a study by Ahmed et al. (2019), major gaps were identified in South Asia, African and Eastern Mediterranean regions on global surveillance of AMR, data collection, sharing and data coordination, method standardization [5]. In recent years, the number of resistant strains has increased, due to causes including an increase in antibiotic usage, inter-generic and inter-specific conjugal transmission of antibiotic-resistant genes between bacteria, and selective pressure [6]. While patterns in AMR can differ within bacterial genera and species, in Bangladesh, Enterobacteriaceae were found to be resistant to the following classes of antibiotics: aminoglycoside, macrolide, and beta-lactams; which was confirmed through analyzing clinical isolates from patients admitted to various tertiary hospitals with acute respiratory infections, wound infections, typhoid fever or diarrhea. In regards to the nature of the genetic aspects of AMR in Bangladesh, the matter is still under observation and the data is limited [7].

Klebsiella pneumoniae (KPN), a member of multidrug-resistant ESKAPE pathogens groups (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) [8], is an agent of both nosocomial and community-acquired infections. Its high association has been found with urinary tract infections, pneumonia, septicemia, burns, pyogenic liver abscesses, wound infections, meningitis, endophthalmitis, and lung abscess [9–13]. KPN strains acquire a multidrug resistant phenotype through horizontal transfer of antimicrobial resistance genes carried by either transposons or plasmids. The transfer is usually mediated by mobile genetic elements, Integrons (IncFII, IncN and IncI1) [14–16]. In Bangladesh, a dramatic increase of β- lactam resistant KPN was observed from 2001 to 2011 [17–19]. The situation became more severe with the discovery and spread of the novel carbapenemase, New Delhi Metallo-ß-lactamase (NDM), proteins that give bacteria the ability to resist multiple types of antibiotics, rendering superbugs. Among 5 variants of NDM (NDM-1 to NDM-5), NDM-1 is endemic in India and also common in South Asian countries such as Pakistan, Nepal and Bangladesh [20–22]. In Bangladesh, NDM-1 has been found significantly prevalent in natural water samples, sewers and even in clinical samples [7, 23, 24].

Because of having a single copy of chromosomal gene bla_SHV-11 that code for SHV-11 and its derivatives which is a class A beta-lactamase, KPN actively and predominantly resists multiple classes of β-lactams [25]. The mutation in the promoter region of the gene, bla_SHV-11 changes the enzyme’s affinity and increases enzyme production or diminishes Penicillin-binding proteins (PBP) located in the cytoplasmic membrane [26–30]. SHV -11 was identified in KPN derived from clinical isolates in Bangladesh [31] and some novel alleles of SHVs were obtained in clinical isolates in Mymensingh. KPN isolates, carrying SHV type ESBL genes were found in natural water samples from lakes in Dhaka [24]. As for the patient samples, isolates from urine and tracheal aspirates contained a prominent distribution of KPN virulence factors. Among those, a capsule associated gene, UDP galacturonate 4-epimerase (uge) is commonly found in KPN and promotes infection by resisting phagocytosis [12]. Surface expression of smooth lipopolysaccharide (LPS) and capsular polysaccharide with K antigen is typically found in wild type KPN isolates. Noteworthy to mention, mutations in the uge gene creates a mutant strain with O-:K- phenotype, lacking a capsule and LPS without O antigen molecules devoid of the outer core oligosaccharide. The mutant strain also transforms UDP-glucose (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) by uridine diphosphate galacturonate 4-epimerases (UDPGLEs) enzymatic activity, rendering a rather avirulent KPN [32, 33].

This study aimed to investigate the antibiotic resistance pattern of KPN in the southern part of Bangladesh through analyzing clinical samples obtained from neonates and adults. We also investigated the prevalence of extended-spectrum beta-lactamases (ESBLs) genes of clinical isolates of MDR KPN in southern Bangladesh.

2 Materials and methods

2.1 Study setting

A cross-sectional study was conducted in two healthcare centers in Chattogram to collect clinical samples including blood, urine, pus, tracheal aspirates, throat swab, umbilical swab, sputum, and wound swab, high vaginal swab (HVS) with traces of KPN from both outdoor and indoor patients. The tenure of sample collection was from August 2018 to November 2019. Samples were collected from various hospital wards such as Medicine, Gynae, Neonatal and Pediatric surgery, Adult surgery, Special care baby unit, Neurology, Diarrhoea, Thalassemia, Orthopedics, Intensive Care Unit (ICU), HDU (Child), Neonatal Intensive Care Unit (NICU). The demographic and epidemiological data of patients with KPN infections were collected from medical records and microbiology databases owned and dispatched by the microbiology laboratory of Chattogram Maa-Shishu O General Hospital Microbiology lab, and CHEVRON hospital and Clinical Laboratory (PTE) Ltd. After screening, we have finally included 1000 samples with confirmed K. pneumoniae (KPN) infections.

2.2 Ethical approval

The study protocol was ethically approved by the institutional review board of Chattogram Maa-Shishu O General Hospital Medical College (Ref: CMOSHMC/IRB/2018/5). Oral consent was obtained from the patients or from parents or guardians (in case of minors). Need for written consent was waived by the ethics committee.

2.3 Confirmation of K. pneumoniae (KPN) isolates

Presence of KPN was confirmed through analyzing microbial cultures, Gram staining and conventional biochemical tests according to CLSI (Clinical Laboratory Standard Institute) guidelines. Initially, the bacterial colony and shape was checked in Mac-Conkey agar medium, Blood agar and Chocolate agar. Gram staining was done for bacterial group differentiation. Once the presence of KPN in clinical samples were confirmed, a number of conventional biochemical tests were performed [Indole, Methyl red, Voges Proskauer, Citrate, TSI (triple sugar Iron, Motility and Urease tests)] using subcultures for of Klebsiella pneumoniae isolates [34–36].

2.4 Determination of antibiotic susceptibility pattern of KPN

The Kirby-Bauer method (KB) was adapted to conduct antibiotic susceptibility tests for KPN obtained from clinical samples. An overnight culture of KPN adjusted to standard suspension of isolate confirming 0.5 McFarland turbidity was inoculated on the surface of two Mueller Hinton agar (HIMEDIA, India) plates using selective antibiotic disks, namely Amikacin (30 μg), Amoxiclav (30 μg), Ampicillin (30μg), Cefepime (30μg), Cefixime (5μg), Ceftazidime (30μg), Ceftriaxone (25μg), Chloramphenicol (30μg), Ciprofloxacin, (5μg), Cotrimoxazole (23.75μg), Gentamicin (10μg), Imipenem (10μg), Levofloxacin (5μg), Meropenem (24μg), Netilmicin (10μg), and Piperacillin (30μg) according to CLSI guidelines [36]. For negative control, a blank disk of filter paper was used. Multidrug resistant samples were selected for molecular analysis. Isolates showing resistance to highest number of antibiotic categories were chosen for further analysis.

2.5 Molecular detection of bla_NDM-1, bla_SHV-11 and uge

The bla_NDM-1, bla_SHV-11 and uge genes from 100 confirmed MDR KPN isolates were identified through polymerase chain reactions (PCR) using the following sets of primers:

bla-_NDM-1: 5’-GGTTTGGCGATCTGGTTTTC-3’; 5’-CGGAATGGCTCATCACGATC-3’ (annealing temperature 51.2°C) [12, 21].
bla_SHV-11: 5’-ATGCGTTATATTCGCCTGTGTATT-3’; 5’-GCGTTGCCAGTGCTCGATCAGCGC-3’ (annealing temperature 51.2°C) [37, 38].
uge: 5’-ATGCGTTATATTCGCCTGTGTATT-3’, 5’-GCGTTGCCAGTGCTCGATCAGCGC-3’ (annealing temperature 52.3°C) [12].

Genomic DNA of KPN was extracted using the Boiling method [39, 40]. Sanger sequencing was carried out with the purified PCR products of a total of 15 isolates (5 isolates from each group carrying bla_NDM-1, blaSHV-11, uge gene). All draft sequences were aligned using BioEdit Sequence Alignment Software (version 7.0.5.3) followed by nucleotide BLAST for further confirmation and to identify nucleotide variations. After analysis, all sequences were deposited to the NCBI database. The accession numbers of SHV-11 gene containing KPN isolates are MN437452, MN551175, MN551176, MN551177 and other sequences have been submitted to NCBI. Before phylogenetic analysis, all obtained nucleotide sequences were translated through the EMBOSS Transeq program. Following translation, the protein sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

2.6 Phylogenetic tree construction

After alignment, a phylogenetic tree was constructed using MEGA7 using the maximum likelihood method [41]. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with a superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

3 Results

3.1 Characteristics of KPN isolates

We analyzed a total of 1000 KPN isolates from two healthcare facilities in this study. The male to female ratio of the participants was found to be1:1.13. By analyzing age groups, children (<15 years age) represented 47.6% of the total study population whereas, young adolescents (15–29 years), adults (30–44 years) and elder age group (≥45 years) covered 18.6%, 10.9% and 22.9%, respectively (Table 1). About 46.5% of the samples were collected from outpatients and 53.5% were from inpatients with KPN infections of which 21.87% were from medicine wards, 19.25% were from the Gynaecology ward, 9.72% were from pediatric surgery wards. Confirmed KPN isolates were collected from a wide variety of samples including urine (39.9%), tracheal aspirates (14.2%), pus (14.1%), blood (13.8%), sputum (10.1%) and others (7.9%) (Table 1). Multidrug resistance was more among the male (74.6%). The prevalence of MDR observed in the urine, tracheal aspiration, blood, pus, sputum and wound swab samples was recorded: 65.1%, 81.25%, 78.2%, 65.9%, 51.4% and 65.9% respectively.

Table 1. Clinical characteristics of study population.

Variable	Total Cases (%) (n = 1000)	Sensitive (%) (n)	MDR (%) (n)	SDR (%) (n)	P value
*Gender*
Male	43% (430)	6.97% (30)	74.65% (321)	18.37% (79)	0.0029
Female	57% (570)	10.87% (62)	63.68% (363)	25.43% (145)
*Age (in years)*
<15 years	47.6% (476)	3.99% (19)	69.12% (329)	26.89% (128)	0.2151
15- <30 years	18.6% (186)	11.83% (22)	70.43% (131)	17.74% (33)
30- <45 years	10.9% (109)	19.27% (21)	62.38% (68)	18.35% (20)
45- <60 years	13.0% (130)	12.31% (16)	69.23% (90)	18.46% (24)
≥ 60 years	9.9% (99)	14.14% (14)	66.67% (66)	19.19% (19)
*Patient type*
Outdoor	46.5% (465)	31.18% (145)	64.73% (301)	25.59% (119)	0.0215
Indoor	53.5% (535)	8.79% (47)	71.58% (383)	19.63% (105)
*Sample types*
Urine	39.9% (399)	12.03% (48)	65.16% (260)	22.81% (91)	<0.001
Tracheal aspirates	14.2% (142)	1.41% (2)	85.21% (121)	13.38% (19)
Pus	14.1% (141)	9.93% (14)	65.96% (93)	24.11% (34)
Blood	13.8% (138)	7.25% (10)	78.26% (108)	14.49% (20)
Sputum	10.1% (101)	11.88% (12)	51.48% (52)	36.63% (37)
Wound swab	4.7% (47)	6.38% (3)	65.95% (31)	27.66% (13)
Others	3.2% (32)	12.5% (4)	56.25% (18)	31.25% (10)

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MDR = Multidrug-resistant, SDR = Single or two drug-resistant

*** P value has been compared between multi drug resistant and single drug resistant samples.

3.2 Antimicrobial resistance patterns of KPN

The majority of KPN isolates were confirmed as multidrug-resistant in the disk diffusion susceptibility test. Most of the isolates were resistant against cefuroxime (79%), cefixime (77%), cefotaxime (74.6%), ceftazidime (71%), cefepime (66%) and ceftriaxone (65%) (Fig 1A) whereas, most effective antibiotics against KPN were amikacin, meropenem and imipenem with the resistance of 26%, 27% and 30%, respectively. Isolates from females showed more resistance to the antibiotics available in the market compared to those from males (Fig 1B).

Fig 1 — (A) Most resistant 10 antibiotics among study samples; (B) Gender wise frequency of antimicrobial resistance in KPN isolates.

Patients were most sensitive to nitrofurantoin (77.6%), amikacin (71.2%) and meropenem (68.6%) (Fig 2A), Isolates from both indoor and outdoor patients were most resistant to cefixime, ceftriaxone and cefepime (Fig 2B).

3.3 Prevalence of bla_NDM-1, bla_SHV-11 and uge among multi-drug resistant KPN isolates

Top 100 multidrug-resistant KPN isolates were selected and screened for determining the prevalence of bla_NDM-1, bla_SHV-11 and uge genes by PCR (Fig 3). 64% of selected KPN isolates were found bla_NDM-1 positive. About 48% of isolates were uge positive and 38% were bla_SHV-11 positive strains (Table 2). A total of 45% of KPN strains having bla_NDM-1 collected from urine samples, 24.2% from tracheal aspirates followed by blood (10.6%), pus (10.6%) and sputum (3%). Most of the patients infected with KPN infections encoding bla_NDM-1 were inpatients (65%), whereas 35% were outpatients (Table 2). The bla_SHV-11 gene was found in urine samples at an increased rate (45%) as compared to tracheal aspiration (27.5%), blood (3.03%), pus (4.5%), throat swab (4.5%), sputum (2%) and wound swab (2%) (Fig 5). The bla_SHV-11 gene was found at an approximately equal ratio in KPN strains of both outpatients (52.7%) and inpatients (47.3%). Similarly, 47% of KPN isolates had uge where 36.2% and 63.8% isolates were from outdoor and indoor patients respectively (Table 2). The uge gene was found in urine (46.8%), tracheal aspirates (27.6%), blood (6.4%), pus (10.6%), sputum (6.4%) (Fig 5). 19 out of 100 isolates contained all three genes bla_NDM-1, bla_SHV-11 and uge. In clinical samples, bla_SHV-11 and uge were more prevalent in males (51.17% and 55.3%, respectively) than in female patients. While bla_NDM-1 gene was prevalent in females (52.3%).

Fig 3 — PCR products were visualized in 1.5% agarose gel using ethidium bromide staining (0.5%). L indicates Ladder (100 bp); NC as a negative control(Nuclease free water); PC indicates positive control, K. *pneumoniae* ATCC70603. ***(A)*** samples representing bands with 621bp were *bla*_NDM-1 positive amplicons similarly, ***(B)*** samples were *bla*_SHV-11 positive amplicons with 858bp, ***(C)*** samples were *uge* positive amplicons having 534bp.

Table 2. Prevalence of bla_NDM-1, bla_SHV-11, uge in top multi-drug resistant KPN samples (n = 100).

Genes	Percentage of positive Isolates	Gender		Patient type
Genes	Percentage of positive Isolates	Male	Female	Outdoor	Wards/Indoor
bla _NDM-1	64	30	34	27	37
uge	47	24	23	17	30
bla _SHV-11	38	21	17	20	18

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For further validation of gene frequency observed in PCR experiments, randomly, 5 PCR amplicons were subjected to gene sequencing. The raw sequences of bla_NDM-1, bla_SHV-11, uge genes were then confirmed by comparing them with the reference sequence database using phylogenetic analysis (Fig 4).

Fig 4 — The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. Three genes are A) NDM-1, B) SHV-11, C) uge.

3.4 Plasmid size determination

All tested isolates were screened for the presence of plasmid. 81 out of 100 isolates were found to have plasmids with a band size of 1500 bp (Fig 6). All plasmids were similar in size which was confirmed by agarose gel electrophoresis.

4 Discussion

Although KPN is a common inhabitant of intestinal microflora, it can metastasize outside the gut triggering a wide range of infections, predominantly in humans. Apart from its metastasizing capacity, what establishes KPN’s reputation as a deadly pathogen is that over time, KPN has been evolving as a superbug. Previous studies have shown that KPN has acquired resistance to carbapenem and other β lactam antibiotics. The results of our study align with the previous studies, in which case, we also found that a high number of KPN isolates showed resistance to cefuroxime, cefixime, cefotaxime, ceftazidime, cefepime, and ceftriaxone. Among total isolates, the majority of the isolates were highly resistant to cefuroxime and the percentage of resistance was higher compared to other antibiotics (Fig 1). A similar finding was observed by Akhter et al. (2016) and Hossain et al. (2017), demonstrating a significant number of Klebsiella isolates resistant to ceftriaxone along with ampicillin and cotrimoxazole [42, 43]. These incidences of resistance were linked to unclean food, contaminated bed sheets, personal hygiene affecting breast-feeding and negligence in washing hands. In all cases, contaminated hands played a significant role in spreading the KPN isolate and promoting its evolution. However, in terms of resistance to broad-spectrum antibiotics, some studies found KPN to be resistant to several more antibiotics apart from the aforementioned [44]. Uddin et al. (2011) observed that Klebsiella spp. were highly resistant to tetracycline, rifampin, nalidixic acid, and 100% sensitive to imipenem. Yasmin et al. (2015) [45] also found that Klebsiella isolates were amoxicillin and nalidixic acid resistant [46]. In contrast, we found the isolates of our study were mostly imipenem, meropenem and amikacin sensitive. We observed an equal distribution of infections in both males and females rin maximum age groups (Table 1). The 50 years male cohort was found to be more prone to the Klebsiella infections [47]. But the incidence of KPN infections was higher among the neonates and children that were included in the study sample. Frequent use of assisted ventilation and extensive administration of antibiotics in first-month post-birth in neonates, naive immune state, immunocompromisation, impairment of gut microflora could be some of the significant causes owing to their susceptibility to KPN infections which led to pulmonary infections and septicemia [42, 48]. Choudhury et al. (2018) stated that 69.2% KPN isolates were the most common gut colonizer in neonates [49]. Considering the overall frequency, the incidence of KPN infections was found higher in patients admitted to the Medicine ward, Gynae ward, Pediatric surgery and neonatal ward among hospitalized patients in our study (Table 1). Urine was found to be the most common reservoir of KPN (39.9%) (Table 1). In a previous study, it was found that KPNs majorly producing ESBLs were isolated from urine samples [43]. Considering our results and that of Hossain et al. (2016), it can be considered that urine is indeed a predominant reservoir for KPN sustenance [43]. Presence of KPN in non-biological samples might be a probable explanation of the phenomenon of horizontal transfer of transposable elements into KPN isolates and rapid transmission of a plasmid to another Enterobacteriaceae [50, 51].

Carbapenem-resistant KPN is considered as a threat worldwide because of the rapid transmission of the plasmid-mediated bla_NDM-1 gene in Klebsiella species and the global spread of NDM-1 producing KPN. According to this study, the scenario is also similar in Chittagong City. In the present observation, 64% of KPN isolates were bla_NDM-1 positive (Fig 2A) (Table 2). NDM-1, belonging to the Class B Metallo-ß-lactamase (MBL) superfamily mediates the catalytic reaction. It has one or two catalytic zinc or iron binds to the active site and cleaves the amide bond of the beta-lactam ring, thus inactivating the antibiotics [52]. The majority of the bla_NDM-1 positive isolates were isolated from female patients than makes (64 vs 34) (Table 2). The incidence of KPN harboring bla_NDM-1 positive isolates was comparatively high in urine (30%) and tracheal aspiration (16%) (Fig 5). Bora and Ahmed (2012) found 8.67% bla_NDM-1 positive KPN isolates in clinical samples in northeast India [53]. 3.5% of NDM-1 producing Enterobacteriaceae was found in clinical samples in the Clinical Microbiology Laboratory of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B) [37]. In another study, 71% of isolates were bla_NDM-1 positive in hospital adjacent areas and 12.1% were positive in the community areas where KPN was prevalent in sewage samples [23]. Colonization of NDM-1 carrying KPN isolates in patients gut and long term hospitalization is also a risk for frequent Klebsiella infections. Plasmid-mediated antibiotic-resistant genes transferred to Klebsiella species or from other gram-negative bacteria or by the transposable elements may be the reasons for the rapid transmission of NDM-1 in environmental samples [20].

The chromosomal bla_SHV-11 was identified in hospitalized and outdoor patients at an equal ratio. Khan et al. (2018) identified 27% isolates producing SHV variants (SHV-201 and SHV-202) from clinical samples. SHV-12 was found in MDR KPN isolates from surface water [24]. The risk factors for the spread of ESBL positive isolates may be prolonged hospitalization, stay in the ICU, invasive entry of ESBL positive isolates through insertion endotracheal, urinary and central venous catheters. In the present study, 48% KPN isolates containing virulence gene, uge were derived and prevalent in urine (54%) and tracheal aspiration (20%). It was similar to the findings of Candan & Aksöz (2015) [12], and defined that there was no correlation among virulence factors and antibiotic-resistant genes, but they both contribute to bacterial pathogenesis. Common sources of KPN and cross-contamination among patients may be the main reasons for the rapid transmission of the uge gene. In our study, 34% KPN isolates were found to harbour NDM-1 and uge in the same strain derived from clinical samples and 19% KPN isolates were defined to carry all tested genes, bla_NDM-1, bla_SHV-11, and uge.

In this study, 81 plasmids of 1400 bps in size were isolated from various clinical samples (Fig 6). A similar result was characterized by Balm et al. (2013), where plasmids of different types and sizes were identified [54]. The size of the plasmids obtained from previous study were ~ 1.4kb in the KPN isolates from urine, sputum samples, and ranged from ~ 9kb to ~ 194kb in blood, tracheal aspirates samples. But another study stated that KPN identified in isolates obtained in the UK, India, and Pakistan had different bla_NDM-1 positive plasmids with different sizes [42]. Islam et al. (2013) reported that harbouring plasmid size ranges from 60 to 100 MDa in KPN and also observed that KPN isolates, causing urinary tract infections to have a plasmid size of 9.8 Kb [20]. The source of plasmid dissemination or spread of KPN in a single hospital or same environment may be the fact of having the same plasmid in KPN isolates. The plasmid-mediated bla_NDM-1 gene and chromosomal bla_SHV-11 showed resistance to carbapenems and fifth-generation of cephalosporins due to the overproduction of NDM-1 enzymes, alteration of penicillin-binding proteins (PBPs), changing cellular permeability and increasing efflux pump [55].

This study found a high prevalence of antibiotic resistance gene bla_NDM-1 along with uge and bla_SHV-11 in KPN strains from isolates in the hospital. Therefore, to obstruct the spread of resistant strains, it is important to take measures for controlling K. pneumoniae infections. In the context of Bangladesh, establishing a national antibiotic resistance surveillance network, scrutinizing susceptibility to carbapenems, determination of clonal relationship based on geographical or epidemiological distribution by pulse-field gel electrophoresis analysis, plasmid conjugation analysis and working with larger samples will allow us to further analyse the patterns of antibiotic-resistant genes in isolates as well as the pool of antibiotics that KPN is resistant to. For further investigations, it is also necessary to analyse the protein products, and other subsidiary cis and trans activating mechanisms associated with bla_NDM-1, bla_SHV-11 and uge.

5 Conclusion

In the K. pneumoniae infection landscape, our study was the first to run an extensive molecular analysis on ESBL producing K. pneumoniae isolates from patients seeking medical care in different hospitals in Chattogram, Bangladesh. Through this study, we were able to identify early occurrences of NDM-1 producing organisms as well as prevalence of blaNDM-1 plasmid. The prevalence of blaNDM-1 plasmid indicates a large-scale distribution of NDM-1 producing bacteria in the community. A substantial increase in resistant KPN strains in the environment, as well as in a healthcare setting, increases the possibility of nosocomial infections; and through horizontal gene transfer, it could also amplify the persistence of the whole antibiotic-resistance situation. Rigorous screening for NDM-1, SHV-11, UGE and other MBLs in ESBL-producing organisms, because it provides insight into a more genotypic aspect on KPN antibiotic resistance along with significant epidemiological factors will help to formulate and implement plans for controlling the use and abuse of antibiotics on a large scale as well as substitute treatment options in resistant cases.

Supporting information

S1 Dataset

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S2 Dataset

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S1 Raw images

(PDF)

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Acknowledgments

The authors would like to thank the research assistants of Disease Biology and Molecular Epidemiology Research Group, Chattogram for their support during the study. We acknowledge support from the Research and Publication Cell, University of Chittagong in this research.

Data Availability

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

Funding Statement

This study was partially funded by Research and publication office, University of Chittagong (Award Number: 6427/Re/Plan/Pub/Div/CU/2018). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

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PLoS One. doi: 10.1371/journal.pone.0257419.r001

Decision Letter 0

Monica Cartelle Gestal

18 Jun 2021

PONE-D-21-08990

Prevalence ‌of‌ ‌antibiotic‌ ‌resistance‌‌ ‌and associated genes among clinical‌ ‌isolates‌ of Klebsiella‌ ‌‌pneumoniae‌ in ‌the southern‌‌ ‌region ‌of‌ ‌Bangladesh

PLOS ONE

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Reviewer #1: The manuscript by Tanni et al. investigated the prevalence of antibiotic resistance and among clinical isolates of K. pneumoniae in Bangladesh. Although I considered the results important under an epidemiological perspective, I regret to say that it presents many weaknesses.

- The are many mistyping through the text

- Resistance nomenclature should also be revised.

- Why did the authors specifically/only targeted the blaNDM-1 and blaSHV-11 genes? Although I recognize that blaNDM-1 is important in this global region, other clinically important ESBL and carbapenemase genes can support the phenotypic results.

- The authors used PCR amplification to identify the blaNDM-1 and blaSHV-11 genes, however more robust data (e.g., resistome, virulome, etc…) could be achieved by using the WGS approach.

- Conclusion sheds light on ESBL, but New Delhi Metallo-β-lactamase-1 (NDM-1) is undoubtedly more important.

Reviewer #2: It is suggested to do a review of the writing, some typographical errors were found. Review the discs used for the sensitivity study. A pie chart might give a better presentation in some cases. There is a duplication when uploading the graphics

Reviewer #3: Dear author,

Please, achieve next suggestions!

Methods:

2.3 Confirmation of K. pneumoniae (KPn) isolates

Please, specify the method used to identify the isolates

Results:

Table 1: Clinical characteristics of study population (n = 1000)

Please, could you divide the age group og 45, into 45-60 and >60. Thanks

Please, improve the figure, the quality and pixeles.

Thanks

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachment

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PLoS One. 2021 Sep 10;16(9):e0257419. doi: 10.1371/journal.pone.0257419.r002

Author response to Decision Letter 0

9 Jul 2021

Response to the reviewer

Reviewer #1:

The manuscript by Tanni et al. investigated the prevalence of antibiotic resistance and among clinical isolates of K. pneumoniae in Bangladesh. Although I considered the results important under an epidemiological perspective, I regret to say that it presents many weaknesses.

Response: The authors would like to thank the reviewer for pointing out the issues and helping us to improve its quality.

- The are many mistyping through the text

Response: we would like to express our apologies for typing errors. In the current format we have checked and corrected the typos.

- Resistance nomenclature should also be revised.

Response: Resistance nomenclature has been changed both by definition and alphabetic order (Page 8 and Table 1). In table-1 a multidrug resistant column has been placed according to the standard definition by AP Magiorakos et al. 2021 and ESPAUR (https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/936199/ESPAUR_Report_2019-20.pdf). In the methodology, names of the antibiotics have been reorganized (Line-167, Page-8).

Response: We would like to thank the reviewer for raising the issue. In this study we have found that the KPN isolates were resistant to more than one class of antibiotics available in the local market, regardless of the age group or type of samples. Then we wanted to find the molecular cause of the resistance in this area which is the second densely populated city in Bangladesh. Based on work that has been done on KPN in Bangladesh, we readily decided to assess the burden of blaNDM-1 and blaSHV-11. Importantly, the prevalence of the blaNDM-1 itself can determine the spread of deadly KPN that is evolving lately. However, for understanding the total scenario an in-depth molecular study is required and we are currently designing to do so.

- The authors used PCR amplification to identify the blaNDM-1 and blaSHV-11 genes, however more robust data (e.g., resistome, virulome, etc…) could be achieved by using the WGS approach.

Response: We agree that the WGS approach is more robust data to confirm the presence of a gene. However, PCR is also a state of the art technique in molecular biology and more convenient to conduct surveillance studies in a resource-limited country like Bangladesh. To check the validity of PCR data, in this study we have randomly checked 15 PCR amplicons by sequencing and found them accurate.

- Conclusion sheds light on ESBL, but New Delhi Metallo-β-lactamase-1 (NDM-1) is undoubtedly more important.

Response: We would like to thank the reviewer for this valuable suggestion. It has been added (Page 20) and conclusion has been modified as-

In the K. pneumoniae infection landscape, our study was the first to run an extensive molecular analysis on ESBL producing K. pneumoniae isolates from patients seeking medical care in different hospitals in Chattogram, Bangladesh. Through this study, we were able to identify early occurrences of NDM-1 producing organisms as well as prevalence of blaNDM-1 plasmid. The prevalence of blaNDM-1 plasmid indicates a large-scale distribution of NDM-1 producing bacteria in the community. A substantial increase in resistant KPN strains in the environment, as well as in a healthcare setting, increases the possibility of nosocomial infections; and through horizontal gene transfer, it could also amplify the persistence of the whole antibiotic-resistance situation. Rigorous screening for NDM-1, SHV-11, UGE and other MBLs in ESBL-producing organisms, because it provides insight into a more genotypic aspect on KPN antibiotic resistance along with significant epidemiological factors will help to formulate and implement plans for controlling the use and abuse of antibiotics on a large scale as well as substitute treatment options in resistant cases.

References:

Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., ... & Monnet, D. L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical microbiology and infection, 18(3), 268-281.

Reviewer #2:

It is suggested to do a review of the writing, some typographical errors were found.

Response: We would like to thank the reviewer for this suggestion. We have rechecked the manuscript and corrected the errors.

Review the discs used for the sensitivity study. A pie chart might give a better presentation in some cases.

Response: We have deleted the information of antibiotics those have been suggested by the reviewers. Moreover, Another chart has been added where the information of sensitivity has been mentioned. A pie chart has been added (Figure 5) where the prevalence of antibiotic resistant genes in different samples has been shown.

There is a duplication when uploading the graphics

Response: Duplicate figures have been removed.

Reviewer #3:

Dear author, Please, achieve next suggestions!

Methods:

2.3. Confirmation of K. pneumoniae (KPN) isolates. Please, specify the method used to identify the isolates.

Response: Initially, the bacterial colony and shape was checked in Mac-Conkey agar medium, Blood agar and Chocolate agar. Then, gram staining was done after culture for bacterial group differentiation. Thirdly, a number of conventional biochemical tests were performed [Indole, Methyl red, Voges Proskauer, Citrate, TSI (triple sugar Iron, Motility and Urease tests)] after subculture for Klebsiella pneumoniae isolates confirmation.This information has been added in the modified manuscript.

First, we checked the bacterial colony and shape in MacConkey agar medium, blood agar, chocolate agar and L.B agar medium. Second, we did gram staining after culture for bacterial group differentiation. Thirdly, some conventional biochemical tests were performed (Indole, Methyl red, Voges Proskauer, Citrate, TSI (triple sugar Iron, Motility and Urease tests) for Klebsiella pneumoniae isolates confirmation. Finally, we selected 100 samples for molecular characterization of resistant determinants (blaNDM-1, blaSHV-11, blauge.). Finally, a phylogram was constructed with the sequence data in comparision with other KPN data retrieved from NCBI.

Results:

Table 1: Clinical characteristics of study population (n = 1000)

Please, could you divide the age group og 45, into 45-60 and >60. Thanks

Response: Thank you for your valuable suggestion. The table has been revised and age group has been divided accordingly (Table 1).

Please, improve the figure, the quality and pixeles. Thanks

Response: Thank you for your valuable suggestion. The figure quality has been improved and the dpi of the modified figure is more than the previous version (300 dpi).

Reviewer’s recommendations

Line 84. When referring to the classes of antibiotics, ESBL is placed, which is a type of resistance and not a class of antibiotic. It is suggested to place beta-lactams as a class of antibiotics

Response: changed ESBL with beta-lactams accordingly.

Line 107. It is not necessary to capitalize Extended

Response: corrected accordingly.

Line 154. It is not necessary to capitalize Standard

Response: amended accordingly.

Lines 155 and 156. It is suggested to place the size of the Petri dishes used since 8 discs should not be placed in boxes of 100 x 10 (15) but a maximum of 6 according to the CLSI recommendations.

Response: We have used 5 or 6 discs in a 90 mm plate. All the recommendations of CLSI (2021) were rechecked and followed. Two of the figures of the study have been attached below (Figure 1).

Figure 1: Disc diffusion test for antibiotic sensitivity assessment.

Line 156. Why do you use Ampicillin disc for Klebsiella? Has natural resistance to this antibiotic

Response: As this is a 600 bed hospital the discs were prepared for the need of all the patients as per requirements for other growths.

Intrinsic resistance to ampicillin is obvious in KPN. Sometimes, KPN carrying beta lactamase genes like blaSHV-11 show different sensitivity patterns to ampicillin (Kim J et al., 2016; Fu Y et al., 2007). Thus, to characterize the sensitivity pattern in different patients we used ampicillin.

Line 157. Why do you place an azithromycin disk? This is used in S.Typhi or Shigella

Response: Herein, we aimed to assess the resistance profile of KPN isolates which were isolated from various clinical and environmental sources against common antibiotics classes. Azithromycin is a bacterial protein synthesis inhibitor of the drug class, Erythromycin and also used for treating intestinal infections. Since, KPN is also an intestinal bacteria we used this disc for susceptibility testing. Moreover, there are previous studies which also mentioned the use of Azithromycin against KPN (Baker et. al. 2019). However, it has been removed from the list and figures of modified manuscript.

The list of antibiotics used for the test does not include any beta-lactamase inhibitor. Why?

Response: We used tazobactam/ piperacillin and amoxicillin/clavulanic acid as beta lactamase inhibitor. We did not mention these in description, because the percentage of resistance or sensitivity was not significant compared to other antibiotics. It has been included in raw data. However, it has also been added to the revised manuscript.

A resistance analysis should be done according to the origin of the patient. Inpatients or outpatients

Response: Resistance for inpatient and outpatient has been analyzed in the revised draft. A figure containing the data has been added in the modified draft (Figure 2, page-26).

Line 214. What was the selection methodology for resistant Klebsiella?

Response: We selected the resistant isolates based on their antimicrobial resistant profile. Multidrug resistant samples were selected for molecular analysis. Isolates showing resistance to the highest number of antibiotics were chosen for further analysis. Isolates that had antibiotic resistant genes were further sent for gene sequencing. It has been added in the modified manuscript (line- 172, page-8).

Line 245, 246. The names of the bacteria must be written in italics