Clonal diversity, virulence factors and antibiotic resistance genes of Enterococcus faecalis and Klebsiella pneumoniae from urban rats in Malaysia

Abstract

Peridomestic rats serve as important carriers of zoonotic pathogens, including multidrug-resistant bacteria such as Enterococcus faecalis and Klebsiella pneumoniae. However, information on the clonal diversity, antibiotic resistance, and virulence of these pathogens in wild urban rats remains limited, particularly in tropical settings. This study investigated E. faecalis and K. pneumoniae in urban rats from wet markets across four Malaysian states. A total of 277 rats (Rattus norvegicus, n = 177; Rattus rattus diardii, n = 100) were trapped, and lung tissues were processed for bacterial isolation. Isolates were identified by 16S rDNA sequencing and examined for antibiotic susceptibility, resistance genes, and virulence factors using PCR. Clonal relationships were determined through multilocus sequence typing (MLST). Nineteen E. faecalis and seven K. pneumoniae isolates were recovered. All E. faecalis isolates carried multiple virulence factors (ace, asa, efaA, and gelE), and four tetracycline-resistant isolates possessed tetL, tetM, and tetS. Five novel E. faecalis sequence types (ST730 - ST734) were identified. All K. pneumoniae isolates carried the SHV gene; two also harbored FOX, and two possessed aerobactin and kfu virulence factors. Two novel K. pneumoniae STs (ST2641 and ST2642) were detected. Phylogenetic analyses revealed that both species displayed diverse sequence types scattered across global lineages. These findings suggest that urban rats may act as potential carriers of virulent and antibiotic-resistant E. faecalis and K. pneumoniae. These findings support the inclusion of urban rats in future One Health surveillance programs investigating antimicrobial resistance and virulent bacterial pathogens.

Highlights

• •Discovered novel sequence types absent from global MLST databases.
• •5 novel STs of E. faecalis and 2 novel STs of K. pneumoniae were discovered.
• •All STs of E. faecalis harbored antibiotic resistance genes (except ST68).
• •All STs of K. pneumoniae harbored antibiotic resistance genes and virulence factors.

1. Introduction

Rats are widely recognized as competent vectors in the transmission of pathogens capable of causing significant human morbidity and mortality, primarily through the contamination of water and food sources [1], [2]. They serve as potential animal carriers for a range of zoonotic pathogens such as Leptospira spp. Rickettsia typhii, Bartonella spp. and Seoul hantavirus, all of which are associated with undifferentiated febrile illnesses in humans, particularly across Asia [1], [3]. These rat-associated diseases are especially prevalent in tropical and subtropical regions, where warm temperatures and high humidity create ideal conditions for pathogen persistence and transmission [1], [3].

Beyond these well-known zoonotic agents, rats may also harbor other high-risk pathogens with serious public health implications. The group of pathogens collectively known as “ESKAPEE” (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) has garnered global attention due to its growing antibiotic resistance and virulence in both healthcare and community settings [4]. While traditionally associated with hospital-acquired infections, recent studies indicate that ESKAPEE pathogens are also present in urban environments, where rats thrive. The potential role of rats as carriers of multidrug-resistant (MDR) ESKAPEE pathogens underscores the urgent need for integrated One Health surveillance approaches to detect and monitor the emergence and potential spread of both community-acquired and nosocomial infections [5]. Wet markets represent unique ecological interfaces where humans, food animals, wildlife, and environmental sources interact closely. Urban rats are frequently observed in these settings due to abundant food sources and may acquire and disseminate bacteria originating from multiple sources. Consequently, surveillance of bacterial pathogens in urban rats may provide useful information regarding the circulation of antimicrobial-resistant and virulent bacteria within these complex ecosystems.

Enterococcus faecalis is commonly found in the environment, including in soil, water, and plant material, and is a natural component of the gastrointestinal microbiota in humans and other mammals, typically at low abundance [6]. Although often commensal, E. faecalis can act as an opportunistic pathogen, particularly in immunocompromised individuals, leading to severe infections such as urinary tract infections (UTIs), bacteremia, endocarditis, and intra-abdominal infections [7]. Similarly, K. pneumoniae is ubiquitous in both environmental and clinical contexts and is found in the human oral cavity, respiratory tract, skin, and gut. Like E. faecalis, K. pneumoniae is a major cause of both community- and hospital-acquired infections, including pneumonia, bacteremia, and UTIs [8], [9]. Both species are also prevalent in animals, where they can cause significant infections leading to multiorgan failure [10], [11]. Importantly, K. pneumoniae carrying clinically relevant extended-spectrum beta-lactamase (ESBL) genes has been detected in urban rat populations in other parts of Asia and West Africa [8], [12], [13]. Schaufler & Nowak [13] suggested that urban rats may contribute to the maintenance and circulation of resistant pathogens, particularly at the human–animal interface, where frequent contact facilitates cross-species transmission.

In Malaysia, a recent local respiratory pathogen surveillance initiative among wild urban rats provided a valuable opportunity to investigate the presence of E. faecalis and K. pneumoniae in this overlooked host species [14]. This work is especially timely, given earlier discoveries of emerging bacteria such as Kocuria marina and Kytococcus schroeteri in urban rats in Malaysia [14], [15], which illustrate the potential of rats to harbor both common and rare pathogenic organisms. To date, no published studies have specifically examined the presence of E. faecalis and K. pneumoniae in urban rats, despite the documented presence of virulent and MDR strains in other animals [16], [17]. The present study provides the first insight into the clonal diversity, antibiotic resistance profiles, and virulence characteristics of E. faecalis and K. pneumoniae isolated from wild urban rats in Malaysia.

2. Materials and methods

2.1. Sample collection and bacterial isolates identification

Rat trappings were conducted at four wet markets, one from each of the following states; Johor (1.4927° N, 103.7414° E), Kedah (6.1248° N, 100.3678° E), Kelantan (6.1168° N, 102.2777° E) and Terengganu (5.3333° N, 103.1500° E) (Fig. 1), at different times in between November 2014 and December 2015. The selected wet markets were located in the respective state capitals and represented the largest wet markets within each state. These sites were selected based on recommendations from the respective local municipal councils, which had reported frequent rodent sightings and rodent-related complaints at these locations. Wet markets were specifically targeted because they provide abundant food resources and opportunities for interactions among humans, animals, and environmental sources, making them appropriate locations for urban rodent surveillance. Steel traps (Length, 28 cm; width, 18 cm; height, 14 cm) baited with salted fish or peanut butter bread were used to capture the rats. Rats were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) [18] prior to species identification using morphological keys [19]. Upon completion of the identification process, euthanasia was performed using an overdose of the same anesthetic agents at three times the initial dosage [18]. Necropsy was subsequently conducted, and lung tissues were aseptically collected and homogenized for bacteriological analysis. Lung tissues were selected because the samples originated from a larger respiratory pathogen surveillance program conducted among urban rats. Internal organs were prioritized to minimize contamination associated with external surfaces and gastrointestinal contents. While E. faecalis and K. pneumoniae are commonly associated with gastrointestinal carriage, their detection in lung tissues may reflect respiratory colonization, systemic dissemination, or opportunistic infection. Surgical tools were changed regularly and the surgical tabletop was sterilized with 70% ethanol in between the processing of each animal to prevent cross over contamination. Lung homogenates from each rat were cultured onto Columbia agar with 5% sheep blood and incubated aerobically overnight at 37 °C. Mixed cultures were subcultured until single colonies were obtained and the colonies were subsequently identified using 16S rDNA sequencing [20]. Isolates identified as E. faecalis and K. pneumoniae were kept in 20% glycerol at −80 °C until further use. The primary objective of the present study was to characterize E. faecalis and K. pneumoniae isolates recovered during pathogen surveillance activities rather than to estimate population-level prevalence. Consequently, all recovered isolates were subjected to further phenotypic and molecular characterization.

Fig. 1.

Locations and coordinates of the respective sampling sites in this study (Johor, Kedah, Kelantan and Terengganu).

2.2. Antibiotic susceptibility tests

Antimicrobial susceptibility profile of E. faecalis isolates and interpretation were performed according to the Clinical and Laboratory Standards Institute (CLSI) M100-S26 guidelines available at the time the laboratory analyses were conducted [21]. The following antibiotic discs were used; ampicillin (10 μg), ciprofloxacin (5 μg), linezolid (30 μg), tetracycline (30 μg) and vancomycin (30 μg) [22]. These antibiotics were selected because they represent clinically important antimicrobial classes commonly used for the treatment of Enterococcus infections and are frequently included in antimicrobial resistance surveillance studies. For K. pneumoniae, ESBL phenotypic confirmatory testing was performed because extended-spectrum beta lactamase production is among the most important resistance mechanisms reported in this species. K. pneumoniae isolates were subjected to the ESBL phenotypic confirmatory test. Antibiotic discs containing cefotaxime (30 μg), cefotaxime-clavulanate (30/10 μg), ceftazidime (30 μg) and ceftazidime-clavulanate (30/10 μg) were used and results were interpreted according to CLSI guidelines [21].

2.3. Detection of antibiotic resistance genes

E. faecalis isolates were screened for the presence of the following genes: tetL, tetM, tetS, gyrA, pbp5 and aac(6′)-aph(2′) [[23], [24]]. The selected antibiotic resistance genes represent commonly reported determinants associated with resistance to glycopeptides, tetracyclines, aminoglycosides, fluoroquinolones, and beta lactams in E. faecalis and have been widely used in molecular epidemiological studies. For K. pneumoniae, the targeted genes included major ESBL families that are frequently associated with clinically significant antimicrobial resistance worldwide [18]. The presence of the following ESBL genes was investigated: CTX-1, CTX-2, CTX-8, CTX-9, CTX-25, OXA-1, OXA-48, TEM, SHV, ACC, FOX, MOX, DHA, CIT, EBC, VEB, GES, PER, VIM, IMP, KPC and NDM. The amplified PCR products were sequenced and compared to the nearest gene homology using BLAST. Primer details are listed in supplementary material S1.

2.4. Detection of virulence factors

Virulence factors for E. faecalis were amplified using established PCR methods [[23], [24]]. Seven genes encoding the collagen-binding protein (ace), aggregation substance (asa), cytolysin activator (cylA), endocarditis antigen (efaA), enterococcal surface protein (esp), gelatinase (gelE) and hyaluronidase (hyl) were tested. PCR was performed to detect virulence factors for K. pneumoniae using primers targeting the mucoviscosity-associated gene (magA), iron siderophore (aerobactin), regulator of mucoid phenotype (rmpA), Klebsiella ferric iron uptake system (kfu) and gene associated with allantoin metabolism (allS) [25], [26]. The amplified PCR products were sequenced and compared with similar genes in the GenBank database. Primer details are listed in supplementary material S1.

2.5. Multilocus sequence typing (MLST)

Seven housekeeping genes of E. faecalis [27] and K. pneumoniae [28] were amplified, sequenced and analyzed according to their respective MLST protocols (https://pubmlst.org/organisms/enterococcus-faecalis and https://bigsdb.pasteur.fr/klebsiella/). Every gene locus was assigned a unique allele number and a sequence type (ST) determined based on the allelic profile from the seven housekeeping genes. The allelic profiles were clustered into minimum spanning trees (MST) using PHYLOViZ Online (https://online.phyloviz.net/index). Primer details are listed in supplementary material S1.

3. Results

The rat trappings conducted in Johor, Kedah, Kelantan and Terengganu (Fig. 1) yielded a total of 277 rats. Morphological identification of the captured rats separated the rats into two distinct species: Rattus norvegicus and Rattus rattus diardii. A total of 177 R. norvegicus were trapped in Johor (n = 19), Kedah (n = 21), Kelantan (n = 99) and Terengganu (n = 38), while 100 R. rattus diardii were trapped in Kedah (n = 36), Kelantan (n = 44) and Terengganu (n = 20).

The recovered bacterial isolates represent opportunistic detections from the sampled rat population and were not intended to estimate pathogen prevalence. Subsequent to 16S rDNA sequencing, 19 E. faecalis isolates were obtained: 10 from Terengganu and nine from Kedah, each isolated from a different rat. No E. faecalis isolates were recovered from rats sampled in Johor or Kelantan. In Terengganu, six isolates were from R. rattus diardii and four from R. norvegicus, whereas in Kedah, five isolates were from R. rattus diardii and four from R. norvegicus. The E. faecalis isolates harbored between two (tetL and tetM) and five (aac(6′)-aph(2′), tetL, tetM and tetS) antibiotic-resistant genes. The gyrA region was successfully amplified and sequenced in isolates TRE158400, TRE157301, and KED152600; however, no mutations associated with fluoroquinolone resistance were identified within the amplified region (supplementary material S2). Two isolates (TRE153500 and TRE152702) did not carry any antibiotic-resistant gene and were classified as ST68 (Table 1). Four Kedah isolates namely KED151401 (ST59), KED152600 (ST202), KED153601 (ST314) and KED155801 (ST474) demonstrated phenotypic tetracycline resistance, and carried all three tested tetracycline resistant genes (tetL, tetM and tetS). Collectively, the E. faecalis isolates carried three to four virulence factors (ace, asa, efaA and gelE), whereas cylA, esp and hyl were not detected. A total of 13 distinct STs were identified across both states, with ST202 detected in both. One E. faecalis isolate could not be determined. Duplicate isolates were found for ST68, ST674 and ST734. Three ST202 isolates were found in Terengganu (n = 2) and Kedah (n = 1). Five STs, (ST730, ST731, ST732, ST733 and ST734) were novel and have been registered in the MLST database. Accession numbers for the amplified amplicons are listed in supplementary material S2.

Table 1.

Characterization of the clonal diversity, antibiotic-resistant genes and virulence factors of E. faecalis from Malaysia.

Isolate name	State	Host	ST	Antibiotic-resistant phenotypec	Antibiotic-resistant gened	Virulence factord
TRE153500	Terengganu	RD	68	–	–	ace, asa, gelE
TRE158400	Terengganu	RD	674	–	tetL, tetM	ace, asa, gelE
TRE155900	Terengganu	RD	ND	–	tetL, tetM	ace, asa, efaA, gelE
TRE158600	Terengganu	RN	730	–	tetL, tetM	ace, asa, efaA, gelE
TRE157002	Terengganu	RN	731	–	tetL, tetM	ace, asa, efaA, gelE
TRE157200	Terengganu	RD	674	–	tetL, tetM	ace, asa, efaA, gelE
TRE157301	Terengganu	RN	732	–	tetL, tetM	ace, asa, efaA, gelE
TRE155201	Terengganu	RD	202	–	tetL, tetM	ace, asa, efaA, gelE
TRE154001	Terengganu	RD	202	–	tetL, tetM	ace, asa, efaA, gelE
TRE152702	Terengganu	RN	68	–	–	ace, asa, efaA, gelE
KED151102	Kedah	RN	192	–	tetL, tetM	ace, asa, efaA, gelE
KED151401	Kedah	RD	59	Tetracycline	aac(6′)-aph(2′), tetL, tetM, tetS	ace, asa, efaA, gelE
KED151001	Kedah	RD	733	–	tetL, tetM	ace, asa, efaA, gelE
KED152201a	Kedah	RN	734	–	tetL, tetM	asa, efaA, gelE
KED152401	Kedah	RN	734	–	aac(6′)-aph(2′), tetL, tetM	ace, asa, efaA, gelE
KED152600	Kedah	RD	202	Tetracycline	aac(6′)-aph(2′), tetL, tetM, tetS	ace, asa, efaA, gelE
KED154601	Kedah	RD	314	Tetracycline	aac(6′)-aph(2′), tetL, tetM, tetS	ace, asa, efaA, gelE
KED155801b	Kedah	RD	474	Tetracycline	aac(6′)-aph(2′), tetL, tetM, tetS	ace, asa, efaA, gelE
KED153201	Kedah	RN	648	–	aac(6′)-aph(2′), tetL, tetM	ace, asa, efaA, gelE

ND, could not be determined; RD, R. rattus diardii; RN, R. norvegicus; ST, sequence type; −, none.

^aThis isolate was recovered together with K. pneumoniae KED152202 from the same rat.

^bThis isolate was recovered together with K. pneumoniae KED155802 from the same rat.

^cStaphylococcus aureus ATCC 25923 and E. faecalis ATCC 29212 were used for quality control for the antibiotic susceptibility test.

^dE. faecalis isolates harboring the various antibiotic-resistant and virulence genes, available in our repository were included as positive controls in the respective PCR amplification runs and sterile water was included as a no template control.

In comparison to E. faecalis, relatively fewer K. pneumoniae isolates were obtained (n = 2 from Terengganu; n = 5 from Kedah,). No K. pneumoniae isolates were recovered from rats sampled in Johor or Kelantan. In Terengganu, K. pneumoniae was isolated from two R. rattus diardii whereas in Kedah, two were from R. rattus diardii and three were from R. norvegicus. All K. pneumoniae isolates carried the SHV antibiotic resistance gene. Isolates TRE153201 and TRE153202 from Terengganu carried an additional FOX antibiotic resistance gene (Table 2). None produced ESBL enzymes. All K. pneumoniae isolates possessed the kfu virulence factor, while TRE153201 and KED154301 additionally carried the aerobactin gene. Six K. pneumoniae STs were identified, with two novel STs (ST2641 and ST2642) discovered in the Terengganu isolates and registered in the MLST database. Four existing STs were found in Kedah (ST37, ST111, ST188 and ST305). Accession numbers for the amplified amplicons are listed in supplementary material S3. Interestingly, two rats yielded both E. faecalis and K. pneumoniae isolates. Isolates KED152201 (E. faecalis) and KED152202 (K. pneumoniae) were recovered from the same rat, as were isolates KED155801 (E. faecalis) and KED155802 (K. pneumoniae), suggesting the possibility of co-colonization by multiple bacterial species within the same host.

Table 2.

Characterization of the clonal diversity, antibiotic-resistant genes and virulence factors of K. pneumoniae from Malaysia.

Isolate name	State	Host	ST	ESBL phenotypec	Antibiotic-resistant gened	Virulence factord
TRE153201	Terengganu	RD	2641	–	SHV, FOX	aerobactin, kfu
TRE153202	Terengganu	RD	2642	–	SHV, FOX	kfu
KED154200	Kedah	RD	111	–	SHV	kfu
KED155802a	Kedah	RN	188	–	SHV	kfu
KED152202b	Kedah	RN	37	–	SHV	kfu
KED153102	Kedah	RD	111	–	SHV	kfu
KED154301	Kedah	RN	305	–	SHV	aerobactin, kfu

RD, R. rattus diardii; RN, R. norvegicus; ST, sequence type; −, none.

^aThis isolate was recovered together with E. faecalis KED155801 from the same rat.

^bThis isolate was recovered together with E. faecalis KED152201 from the same rat.

^cE. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality control for the ESBL phenotypic confirmatory test.

^dK. pneumoniae isolates harboring the various antibiotic-resistant and virulence genes, available in our repository were included as positive controls in the respective PCR amplification runs and sterile water was included as a no template control.

The MST analysis demonstrated that the identified E. faecalis STs were widely distributed across the global population structure and did not cluster into a single lineage, indicating substantial genetic diversity (Fig. 2). Several STs were closely related to lineages previously reported from clinical, livestock, and environmental sources, while five novel STs (ST730–ST734) were identified. Similarly, the identified K. pneumoniae STs were dispersed throughout the global K. pneumoniae population structure, indicating considerable genetic diversity (Fig. 3). Two novel STs (ST2641 and ST2642) were identified alongside lineages previously reported from human and animal sources.

Fig. 2.

The MST generated using PHYLOViZ Online illustrates that the STs of E. faecalis are widely scattered globally, indicating high genetic diversity.

Note: Nodes – STs; blue boxes – STs that were found in the present study; black peripheral boxes - the exact position of the STs and their genetic linkages to the parent ST; red nodes - the parent STs; green nodes - the STs that were classified in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

The MST generated using PHYLOViZ Online illustrates that the STs of K. pneumoniae are widely scattered globally, indicating high genetic diversity.

Note: Nodes – STs; blue boxes – STs that were found in the present study; black peripheral boxes - the exact position of the STs and their genetic linkages to the parent ST; purple boxes - the major K. pneumoniae clonal complexes.; red nodes - the parent STs; green nodes - the STs that were classified in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

E. faecalis is a significant cause of bacteremia, with mortality rates reaching up to 20%, and commonly requiring antibiotics such as ampicillin or vancomycin, often paired with ceftriaxone or gentamicin [29]. In this study, all tetracycline-resistant E. faecalis isolates carried tetL, tetM, and tetS, whereas isolates lacking tetS were not phenotypically resistant to tetracycline. However, because only disc diffusion susceptibility testing was performed and no minimum inhibitory concentration (MIC) data were generated, the specific contribution of tetS to the resistance phenotype could not be determined. Misuse of antibiotics in poultry production may introduce antibiotic residues in poultry products [30], supporting the possibility that rodents in environments such as wet markets acquire resistant strains through the contaminated food chain. Although four tetracycline-resistant E. faecalis isolates carried the aminoglycoside resistance gene, aac(6′)-aph(2′), none displayed phenotypic resistance to gentamicin. This highlights a critical concern, as resistance genes, despite lacking current phenotypic expression, can be transferred horizontally to unrelated bacterial pathogens, facilitating the widespread emergence of antimicrobial resistance [31]. Peridomestic rats are increasingly recognized as potential carriers of antibiotic-resistant bacteria, reflecting environment contamination [32].

The presence of virulence factors in these E. faecalis isolates suggests their potential to cause severe infections in humans [23]. The detected virulence factors included ace (collagen-binding adhesin); asa (aggregation substance promoting bacterial clumping and biofilm formation); efaA (adhesin); and gelE (gelatinase capable of degrading host extracellular matrix components). These virulence factors play key roles in the pathogenesis of infective endocarditis, enabling bloodstream entry, adherence to host tissues and induce tissues inflammation. Although targeted studies on E. faecalis in rats are limited, the detection of E. faecalis carrying multiple virulence factors in rodent lung homogenates further underscores their potential as environmental carriers for virulence and MDR strains [[6], [23]].

The recovery of identical sequence types (ST68, ST674, ST734, and ST202) from multiple rats suggests that certain E. faecalis lineages may be circulating locally within urban rat populations, although the limited number of isolates precludes firm conclusions regarding transmission dynamics. The broad distribution of E. faecalis STs across the global population structure highlights the considerable genetic diversity of isolates recovered from urban rats. Notably, five novel sequence types (ST730–ST734) were identified, representing previously unreported E. faecalis lineages and expanding the current MLST database. Several of the identified STs were closely related to lineages belonging to the globally distributed CC2/CC6 complexes, which have previously been associated with healthcare-associated infections and dissemination across multiple countries [[22], [33], [34]]. Among these, ST59 is of particular interest because MDR strains have been reported from both human and animal sources [[35], [36], [37]]. In the present study, ST59 exhibited tetracycline resistance and carried multiple resistance determinants. Furthermore, ST202 was recovered from multiple rats and has previously been reported from hospital-associated specimens, food, animal, and environmental settings, highlighting its ecological versatility [[22], [33], [36], [38]]. The widespread occurrence of E. faecalis lineages carrying tetracycline resistance determinants highlights the persistence of antimicrobial resistance across diverse ecological settings [[22], [36], [39]]. Wet markets represent complex environments where humans, animals, food products, and environmental sources interact closely, making them useful locations for pathogen surveillance and monitoring of antimicrobial resistance. Although two cases of simultaneous recovery of E. faecalis and K. pneumoniae were observed, this finding may be epidemiologically relevant because urban rats are exposed to diverse microbial communities and may act as carriers of multiple bacterial species. The co-occurrence of bacteria harboring antibiotic resistance and virulence determinants within the same host could potentially contribute to the maintenance and environmental dissemination of these traits. However, the limited number of observations and the absence of investigations into bacterial interactions or horizontal gene transfer preclude definitive conclusions. Further studies are required to determine the frequency and epidemiological significance of such co-colonization events.

Compared with E. faecalis, K. pneumoniae demonstrated substantially greater genetic diversity, with more than 8000 STs currently recorded in the Pasteur MLST database. The K. pneumoniae STs identified in this study have previously been reported from human, animal, food, and environmental sources, reflecting the broad ecological distribution of this species [[17], [40], [41], [42], [43]]. Particularly noteworthy was the discovery of two novel sequence types, ST2641 and ST2642, which expanded the known diversity of K. pneumoniae associated with urban rats. Although ST2642 appears related to the clinically important ST23 lineage and ST2641 shares ancestry with the ST292 lineage (Fig. 3), further genomic investigations would be required to determine whether these novel STs possess similar virulence or antimicrobial resistance characteristics [[12], [44], [45]].

K. pneumoniae was recovered infrequently in this study; however, all isolates carried the SHV beta lactamase gene and two additionally harbored the FOX gene. Furthermore, all isolates possessed the kfu virulence factor, while two also carried aerobactin. Both virulence factors have been associated with enhanced iron acquisition, increased invasiveness, and hypervirulence in K. pneumoniae [[9], [46]]. Collectively, these findings demonstrate the presence of clinically relevant virulence and resistance determinants among K. pneumoniae circulating in urban rat populations and highlight the value of continued surveillance [47].

This study has several limitations. First, only one wet market was sampled in each state. Although these markets were selected because they were the largest wet markets in their respective state capitals and had frequent reports of rodent activity, the findings may not be representative of all wet markets or urban rat populations within the respective states. Second, although 277 rats were examined, only 19 E. faecalis and seven K. pneumoniae isolates were recovered. Consequently, the findings should be interpreted as characterization of recovered isolates rather than estimates of pathogen prevalence or transmission risk within urban rat populations. Third, the relatively limited number of isolates and restricted geographic coverage may not fully represent the true diversity of E. faecalis and K. pneumoniae circulating among urban rats in Malaysia. Fourth, funding constraints limited the scope of laboratory investigations, including the range of antibiotics tested and the ability to sample additional locations. These factors may have influenced the overall detection rates and potentially underestimated the true burden of antimicrobial resistance and virulent strains. Fifth, the present study utilized lung tissues collected as part of a respiratory pathogen surveillance program. Although E. faecalis and K. pneumoniae are commonly associated with gastrointestinal and mucosal carriage, their recovery from lung tissues remains noteworthy and may reflect respiratory colonization, opportunistic infection, or systemic dissemination in urban rats. Nevertheless, the exclusive use of lung tissues limits conclusions regarding intestinal carriage and environmental circulation. Future studies incorporating intestinal, fecal, and environmental samples would provide a more comprehensive understanding of the ecology and transmission potential of these bacteria.

5. Conclusions

This study provides the first insight into the clonal diversity, antibiotic resistance profiles, and virulence factors of E. faecalis and K. pneumoniae from wild urban rats in Malaysia. The limited occurrence of phenotypically resistant E. faecalis and non-ESBL-producing K. pneumoniae suggests limited antibiotic selection pressure in their natural environment. Nevertheless, the detection of virulence-associated genes and globally relevant STs highlights the occurrence of clinically important pathogens in urban rat populations. These findings support the inclusion of urban rats in future One Health surveillance programs investigating antimicrobial resistance and bacterial pathogen circulation.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work the authors used ChatGPT (OpenAI, GPT-4) to assist with language refinement and grammatical editing of the manuscript. The authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

CRediT authorship contribution statement

Phui Chyng Yap: Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis. Norhidayu Sahimin: Writing – review & editing, Validation, Supervision, Resources, Investigation, Funding acquisition, Data curation. Nurul Asma Anati Che-Mat-Seri: Investigation, Formal analysis, Data curation. Intan Noor Aina Kamaruzaman: Methodology, Investigation, Formal analysis. Morsid Andityas: Methodology, Investigation, Formal analysis. Hai Yen Lee: Writing – review & editing, Validation, Formal analysis. Siti Nursheena Mohd-Zain: Resources, Project administration, Funding acquisition. Sazaly AbuBakar: Writing – review & editing, Validation, Supervision, Resources, Funding acquisition. Shih Keng Loong: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Ethical approval

All procedures performed in this study which involves animals were carried out in compliance to the guidelines, ethical standards and recommendations of the Universiti Malaya Institutional Animal Care and Use Committee (UM IACUC). This study was approved by the UM IACUC with the ethics reference no. ISB/31/01/2013/SNMZ (R) and did not involve any endangered or protected animal species.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Appendix A. Supplementary data

Supplementary material

Data availability

All data supporting the findings of this study are included within the manuscript and its Supplementary Materials. The nucleotide sequences generated in this study, including 16S rDNA, antibiotic resistance genes, and virulence factors, have been deposited in the NCBI GenBank database. The corresponding accession numbers are provided in Supplementary Tables S2 and S3. The MLST data and novel STs for E. faecalis and K. pneumoniae have been deposited into and can be accessed from their respective databases (https://pubmlst.org/organisms/enterococcus-faecalis and https://bigsdb.pasteur.fr/klebsiella/).