Urban Cockroaches as Reservoirs of Multidrug-Resistant Bacteria: Insights from Morphological and Molecular Characterization

ABSTRACT

Cockroaches are recognized as important mechanical vectors of antimicrobial-resistant bacteria in urban environments. This study examined the diversity of cockroach species, seasonal abundance, and associated bacteria across four urban biotopes, i.e., households, warehouses, hospitals, and canteens. We hypothesized that cockroach diversity and bacterial resistance profiles would differ significantly by sub-division, season, and habitat type, reflecting environmental and anthropogenic influences. Sampling was conducted by handpicking and using baited traps. Bacteria from the gut and surface were characterized using morphological, biochemical, and 16S rRNA sequencing. Resistance was tested using the Kirby–Bauer disk diffusion method against eight antibiotics, including azithromycin, ciprofloxacin, levofloxacin, cefixime, ceftriaxone, amoxicillin, tetracycline, and cefaclor. Four cockroach species were recorded; Blattella germanica and Periplaneta americana were the most widespread, while Pycnoscelus surinamensis occurred only in Wazirabad. Households showed the highest diversity (Simpson’s 1–D = 0.573; Shannon’s H = 0.939), and summer had the greatest richness and diversity (1–D = 0.565; H = 0.919), whereas winter had the lowest. Cefixime resistance was the highest (90%), while ciprofloxacin and levofloxacin had the lowest (5%). Multidrug resistance occurred in 35% of strains, 60% were resistant to at least one antibiotic class, and one strain was pan-susceptible. Seventeen of twenty strains (85%) had a Multiple Antibiotic Resistance (MAR) index >0.2, indicating a significant public health risk. The seasonal and habitat-related variations in both cockroach abundance and bacterial resistance patterns underscore their role in the spread of multidrug resistant bacteria. Integrated pest management and targeted antibiotic stewardship are essential to mitigate this emerging urban health threat.

Keywords:
Cockroach species; Gut microbiota; Antimicrobial resistance; Multidrug resistance; Public health risk

Introduction

Cockroaches, belonging to the order Blattodea, comprise more than 4,600 described species worldwide (Memona et al., 2017; Guzman and Vilcinskas, 2020). While most species are harmless, a few, such as Periplaneta americana, Blatta orientalis, Blattella germanica, and Supella longipalpa, have become notorious household pests (Chompoosri et al., 2004; Memona et al., 2017). These pests thrive in human dwellings owing to their omnivorous feeding habits, tolerance for unsanitary conditions, and preference for warm, humid environments (Mullins, 2015). Commonly found in kitchens, hospitals, garbage areas, and sewers, pose considerable public health risks by contaminating food and disseminating pathogenic microorganisms (Graczyk et al., 2005; Bouamamaa et al., 2010; Nasirian, 2017).

In addition to being nuisance pests, cockroaches are recognized carriers of pathogenic microorganisms, with their occurrence linked to allergies, foodborne illnesses, and hospital-acquired infections (Peterson and Shurdut, 1999; Turner et al., 2022). Both the gut and body surfaces can harbor multidrug-resistant (MDR) bacteria such as Escherichia coli, Salmonella spp., Klebsiella spp., Staphylococcus aureus, and Pseudomonas aeruginosa (Naher et al., 2018; Kabantiyok et al., 2023). Reports suggest that up to 90% of gut-associated bacteria in cockroaches are MDR, with Gram-positive strains showing particularly high resistance (Solomon et al., 2018).

Their presence in human-inhabited environments represents a substantial health risk due to the increased likelihood of transmitting MDR bacteria (Soureshjani and Doosti, 2013). They may contribute to the spread of foodborne diseases and allergic reactions (Patel and Meher, 2016; Nasirian, 2019; Wang et al., 2019), and are known vectors of agents causing diseases such as plague, dysentery, abscesses, and leprosy (Adedara et al., 2022). Over 100 bacterial species have been documented in symbiotic association with cockroaches (Jalil et al., 2012). Evidence also suggests their potential to mechanically transfer viruses, including SARS-CoV-2, through contact with contaminated fecal matter (Sharawi, 2021).

Being nocturnal and regularly shedding their cuticle, cockroaches frequently encounter contaminated materials that enhance their efficiency as disease transmitters (Fischer et al., 2003; Mpuchane et al., 2006). Particularly concerning is their role in harboring and spreading antimicrobial-resistant bacteria, especially in healthcare settings. Studies have reported resistance to widely used antibiotics, including amoxicillin, vancomycin, and chloramphenicol, among bacteria isolated from cockroaches (Zarchi and Vatani, 2009; Kundera et al., 2020), though some strains remain susceptible to agents such as ciprofloxacin and imipenem (Ghasemi-Dehkordi et al., 2016).

Despite global recognition of this issue, a notable research gap exists in Pakistan. While some national studies have examined AMR in insects (Bilal et al., 2021; Kamran et al., 2021), few have specifically investigated cockroach-associated bacteria in urban environments, and no comprehensive, habitat-wide survey has been conducted in District Gujranwala. This densely populated and industrialized city in Punjab, Pakistan, offers favorable conditions for cockroach proliferation, including summer temperatures ranging from 36 to 42 °C, poor sanitation, and abundant food waste (Baig and Ehrenreich, 1997). We hypothesized that cockroach populations in Gujranwala exhibit species-specific seasonal abundance patterns influenced by local habitat conditions, and that these populations harbor diverse cultivable bacterial communities with varying levels of antimicrobial resistance.

Accordingly, this study aimed to identify the predominant cockroach species present in households, warehouses, hospitals, and canteens, isolate and characterize bacteria from both their gut and external surfaces, and assess the antimicrobial resistance of these isolates to eight commonly used antibiotics using the Kirby–Bauer disk diffusion method (Malinga et al., 2022). To confirm bacterial identity and explore their evolutionary relationships, 16S rRNA gene sequencing was performed. By integrating data on cockroach species diversity, seasonal abundance, and resistance profiles, this research provides valuable insights into their role as reservoirs and vectors of multidrug-resistant pathogens, with important implications for public health surveillance and control strategies.

Materials and methods

Study area

Gujranwala (32.1877°N, 74.1945°E; 231 meters above sea level), is an industrial city in Punjab, Pakistan, characterized by a hot semi-arid climate (BSh) with summer temperatures averaging 36°C to 42°C and winter temperatures averaging around 7°C (Baig and Ehrenreich, 1997). The district consists of five administrative subdivisions and has an area of 3,198 km², and lies at an elevation of 744 feet above sea level (Mahmood et al., 2013). The monsoon season peaks in July and August, which are also the rainiest months (Faheem et al., 2007; Mahmood et al., 2013).

Sampling methodology

Sampling was conducted across five sub divisions of District Gujranwala: Gujranwala City, Gujranwala Saddar, Kamoke, Wazirabad, and Nowshera Virkan. Four urban biotopes were selected: households, warehouses, hospitals, and canteens. Cockroach collection occurred fortnightly from August 2020 to July 2021. Active sampling was performed manually for approximately three hours per day during early mornings and at night to coincide with peak cockroach activity (Mehainaoui et al., 2021). Passive sampling involved placing 10 baited traps in each biotope per subdivision every two weeks and leaving them overnight. Trap spacing ranged from 10 to 50 ft, depending on site layout and accessibility.

Traps were placed in known cockroach-harboring areas, including kitchen cupboards, washrooms, and behind sinks in households; trash areas, drains, sinks, and food preparation zones in canteens; cafeterias, storage areas, and plumbing zones in hospitals; and corridors and damp, concealed areas in warehouses.

Identification of cockroach specimens

The collected specimens were identified to species level using published taxonomic keys and entomological literature specific to cockroaches (Hagenbuch et al., 1988; Choate et al., 2008).

Bacterial isolation from the Surface and gut

To investigate bacterial communities, four adult cockroaches were collected from each habitat type within each subdivision, except Pycnoscelus surinamensis, which was only found in household settings in Wazirabad. The live specimens were immobilized by placing them at 0 °C for 15 minutes. To isolate surface-associated bacteria, each cockroach was vigorously shaken in 200 µl of 2% saline solution, and the resulting suspension was subjected to two serial dilutions using autoclaved distilled water (Khan et al., 2018). For gut-associated bacteria, cockroaches were surface-sterilized by immersion in 70% ethanol for 5 min, rinsed with 2% saline, and air-dried under a laminar hood (Liu et al., 2016). Under sterile conditions, the alimentary canal was dissected and macerated, and six serial dilutions were prepared. Aliquots (0.02 ml) from both surface and gut samples were inoculated onto nutrient agar plates and incubated at 37 °C overnight (Ofoedu et al., 2021).

Morphological and biochemical characterization

Bacterial isolates were initially characterized by colony color, shape, and margin. Biochemical tests included catalase, citrate utilization, and indole, following established protocols (Garrity and Holt, 2001). Bubble formation indicated catalase positivity; green-to-blue color change indicated citrate utilization; and a red ring indicated an indole-positive reaction. Isolates were stored in 15% glycerol at −20 °C.

Bacterial DNA extraction

Genomic DNA was extracted from isolates using the boiling lysis method. A 200 µl aliquot of bacterial broth was centrifuged at 10,000 rpm for 2 minutes. The pellet was washed twice with distilled water. The cell pellet was then boiled for 12–15 minutes to lyse the cells, and the supernatant containing DNA was collected post-centrifugation for use in PCR.

16S rRNA gene amplification, sequencing, and phylogenetic analysis

The primer pair used in this study included the 16F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1510R (5′- GGCTACCTTGTTACGA-3′), which were utilized to get an amplification product of 1500 bp (Weisburg et al., 1991). PCR reactions contained 1× DreamTaq™ Green PCR Master Mix (12.5 µl), 10 µM of each primer, 5 µl DNA template, and nuclease-free water to 25 µl total volume (Máca et al., 2021). Cycling conditions: initial denaturation at 94 °C for 5 min; 30 cycles of 95 °C for 45s, 50 °C for 30 s, and 72 °C for 10 min; final extension at 72 °C for 5 min (Christopher et al., 2021). Products were visualized by agarose gel electrophoresis. Sanger sequencing was performed commercially (Lab Genetics, Lahore). Sequences were edited using CHROMAS 2 software and aligned via BLAST against the NCBI database. Phylogenetic relationships were analyzed using MEGA 6.0 and the Neighbor-Joining method with bootstrap values based on 1000 replicates (Tamura and Nei, 1993; Kumar et al., 2018; Javaid et al., 2021).

Phylogenetic relationships of bacterial strains isolated from the gut and body surface of urban cockroaches based on 16S rRNA sequences were analysed by decoding the strain codes. Strain codes indicate the source, cockroach number, and colony morphology of each bacterial isolate.

Antibiotic Susceptibility Testing and AMR analysis

The antimicrobial resistance (AMR) profiles of the bacterial isolates were evaluated using the Kirby-Bauer disk diffusion method (Varaldo, 2002). The antibiotics tested included: azithromycin (AZM, 15 µg), ciprofloxacin (CIP, 5 µg), levofloxacin (LEV, 5 µg), cefixime (CFM, 5 µg), ceftriaxone (CRO, 30 µg), amoxicillin (AML, 10 µg), tetracycline (TE, 30 µg), and cefaclor (CEC, 30 µg), all from Oxoid UK.

Bacterial suspensions were adjusted to a 0.5 McFarland standard to standardize inoculum density (Jacobs and Chenia, 2007). Nutrient agar plates were poured to a depth of 4 mm as recommended by CLSI (2020). Plates were dried at room temperature for 3–5 minutes before placing antibiotic discs approximately 24 mm apart using sterile forceps.

Inhibition zones were measured in millimeters and interpreted as resistant, intermediate, or susceptible based on CLSI guidelines (Weinstein and Lewis 2nd, 2020). The Multiple Antibiotic Resistance (MAR) index was calculated as the ratio of the number of antibiotics to which an isolate was resistant over the total number tested. Strains were categorized as multidrug-resistant (MDR) if they were resistant to at least one agent in three or more antibiotic classes (Magiorakos et al., 2012).

Statistical analysis

Data were compiled based on monthly sampling conducted across five sub divisions of Gujranwala District, Pakistan. The seasons were categorized as Summer (May–July), Spring (March–April), Autumn (August–October), and Winter (November–February). Species diversity within each season, habitat, and biotype was assessed using the Shannon-Wiener diversity index and Simpson’s diversity index. These indices were computed using PAST. To examine how ecological factors influence cockroach community composition, two separate two-way PERMANOVA tests were performed using PAST Software (v4.03). Analyses were based on a Bray-Curtis similarity matrix derived from species abundance data, with 9,999 permutations for statistical robustness (Dallarés et al., 2017). The first test assessed the effects of season (summer, autumn, winter, spring) and location (five sub divisions), while the second evaluated biotype (household, warehouse, hospital, canteen) and location. A significance threshold of p < 0.05 was applied.

For phylogenetic analysis of bacterial isolates, 16S rRNA gene sequences (~1500 bp) were aligned, and evolutionary relationships were inferred using the Neighbor-Joining (NJ) method in MEGA version 6.0. Bootstrap analysis with 1000 replicates was performed to assess the robustness of branching nodes. The scale bar in the phylogenetic tree indicates the number of base substitutions per site. Sequence-based clustering patterns were used to infer relatedness among isolates and to validate taxonomic identification obtained through BLAST analysis.

Results

Diversity and relative abundance of cockroaches

A total of 2895 cockroach specimens were collected from various urban biotopes in District Gujranwala. These specimens represented four species: Blattella germanica (Family: Ectobiidae, formerly Blattellidae), Periplaneta americana (Family: Blattidae), Blatta lateralis (Family: Blattidae), and Pycnoscelus surinamensis (Family: Blaberidae). Among the collected specimens, Blattella germanica was the most dominant species, followed by Periplaneta americana. The relative abundance of cockroach species varied across biotypes and sub divisions. Blattella germanica and Periplaneta americana were present across all biotypes, with the highest abundances observed in households. Blatta lateralis was also most common in households, particularly in Kamonke (77.78%) and Gujranwala Saddar (57.78%). Pycnoscelus surinamensis was exclusively recorded in Wazirabad, comprising 14.29% of canteen and 85.71% of household collections. Overall, households consistently exhibited the highest species abundances across all sub divisions (Table 1).

Table 1
Relative abundance (%) of four cockroach species across different biotypes (household, warehouse, hospital, canteen) within five sub divisions of Gujranwala District.

Subdivisions	Biotypes	B. lateralis	B. germanica	P. americana	P. surinamensis
Gujranwala City	Canteen	13.89	22.26	19.56	--
	Hospital	8.33	16.61	10.67	--
	Household	44.44	33.89	43.11	--
	Warehouse	33.33	27.24	26.67	--
Gujranwala Saddar	Canteen	8.89	22.06	18.87	--
	Hospital	20.00	23.06	21.13	--
	Household	57.78	33.08	38.03	--
	Warehouse	13.33	21.80	21.97	--
Kamonke	Canteen	5.56	18.23	12.03	--
	Hospital	--	14.29	18.99	--
	Household	77.78	43.84	47.47	--
	Warehouse	16.67	23.65	21.52	--
Nowshera Virkaan	Canteen	6.25	19.57	17.32	--
	Hospital	18.75	16.09	14.53	--
	Household	56.25	43.91	47.49	--
	Warehouse	18.75	20.43	20.67	--
Wazirabad	Canteen	28.21	27.04	29.39	14.29
	Hospital	10.26	12.68	13.42	--
	Household	56.41	46.48	46.96	85.71
	Warehouse	5.13	13.80	10.22	--
Overall	Gujranwala City	21.18	20.23	18.29	--
	Gujranwala Saddar	26.47	26.81	28.86	--
	Kamonke	10.59	13.64	12.85	--
	Nowshera Virkaan	18.82	15.46	14.55	--
	Wazirabad	22.94	23.86	25.45	100.00

Overall, diversity and dominance indices showed minor variation, suggesting a broadly similar cockroach community structure across locations. Summer displayed the highest diversity and abundance, while winter showed the lowest diversity and greatest dominance. Shannon diversity index (H') was highest in summer (0.928) and lowest in winter (0.686), indicating more even distribution and richness in summer. Simpson's diversity (1-D) ranged from 0.565 (summer) to 0.4668 (winter), supporting the same trend. Cockroach diversity varied across biotypes. Households had the highest individual abundance (n = 1230) and highest diversity, reflected by the Shannon index (0.939) and Simpson’s 1-D (0.573). Canteens and hospitals showed moderate diversity, with Taxa_S = 4 and 3, respectively (Table 2). A two-way PERMANOVA (9,999 permutations) revealed a significant effect of season on cockroach community composition (F = 28.07, p = 0.0001), and a weaker but significant effect of location (F = 2.22, p = 0.0388). The season × location interaction was not significant (F = 1.02, p = 0.4362), indicating consistent seasonal patterns across sites.

Table 2
Diversity indices of cockroach communities across different sub divisions, biotypes, and seasons in Gujranwala District.

Category Type	Description	Taxa_S	Individuals	Simpson (1–D)	Shannon_H	Evenness (e^H/S)
Subdivisions	Gujranwala City	3	562	0.549	0.877	0.801
	Gujranwala Saddar	3	799	0.550	0.869	0.795
	Kamonke	3	379	0.537	0.844	0.775
	Nowshera Virkaan	3	441	0.558	0.896	0.817
Biotype	Canteen	4	610	0.529	0.830	0.573
	Hospital	3	472	0.536	0.842	0.774
	Household	4	1230	0.573	0.939	0.640
	Warehouse	3	583	0.538	0.848	0.779
Season	Autumn	3	691	0.539	0.848	0.778
	Spring	3	324	0.537	0.847	0.777
	Summer	4	1712	0.565	0.919	0.627
	Winter	3	168	0.467	0.686	0.662

In contrast, a second two-way PERMANOVA, assessing biotype and location, showed no significant effects of biotype (F = 2.09, p = 0.0669), location (F = 0.98, p = 0.4488), or their interaction (F = 0.22, p = 1.000), suggesting similar species composition across these categories.

Colonial morphology and biochemical characterization

Twenty bacterial strains isolated from the gut and surface of cockroaches were characterized based on colony morphology, specifically color, shape, and margin, and were assigned strain codes accordingly (Table 3). Most isolates were catalase positive, with only a few exceptions that showed no or weak effervescence. Similarly, the majority of strains exhibited positive citrate utilization, indicated by a color shift from green to blue, while a limited number remained negative. Indole production was observed in five strains, which turned red upon testing, whereas indole-negative strains showed no color change or turned yellow (Figure 1). These biochemical traits aided in a preliminary differentiation of the bacterial isolates before molecular identification.

Table 3
MAR Index calculation for all tested bacterial strains against eight antibiotics used.

Strain ID	Description	Number of Resistant Antibiotics	MAR Index
G1CF	Gut 1 – Creamy Filamentous	6	0.75
G1YR	Gut 1 – Yellow Round	4	0.50
G2RT	Gut 2 – Round Transparent	3	0.38
G2CR	Gut 2 – Creamy Round	3	0.38
G2WR	Gut 2 – White Round	0	0.00
G4OC	Gut 4 – Off-white Curled	3	0.38
G4CF	Gut 4 – Creamy Filamentous	1	0.13
G4WC	Gut 4 – White Curled	5	0.63
G5YCF	Gut 5 – Yellowish Creamy sc Filamentous	3	0.38
G5YRW	Gut 5 – Yellowish Round Whitish	6	0.75
G5WLI	Gut 5 – White Large Irregular	4	0.50
G6CR	Gut 6 – Creamy Round	1	0.13
G6PYF	Gut 6 – Pale Yellow Filamentous	3	0.38
S1WF	Surface 1 – White Filamentous	6	0.75
S1CF	Surface 1 – Creamy Filamentous	4	0.50
S2WF	Surface 2 – White Filamentous	5	0.63
S2CC	Surface 2 – Creamy Curled	2	0.25
S2CF	Surface 2 – Creamy Filamentous	2	0.25
S3WR	Surface 3 – Whitish Round	5	0.63
S4WIC	Surface 4 – White Irregular Curled	2	0.25

Figure 1
Biochemical characterization of bacterial isolates: (A) Catalase test, (B) Citrate Utilization test, (C) Indole Test.

Antimicrobial susceptibility

Antibiotic susceptibility testing revealed notable resistance trends among the twenty bacterial strains tested against eight antibiotics (Figure 2). Seventeen out of twenty strains exhibited a Multiple Antibiotic Resistance (MAR) index greater than 0.2, indicating exposure to high-risk contamination sources (Table 3). In total, 35% (7/20) of the strains were classified as multidrug-resistant (MDR), showing resistance to at least three different antibiotic classes. An additional 60% (12/20) of the strains were resistant to at least one antibiotic class, while only one strain (G2WR) was pan-susceptible, exhibiting no resistance to any of the antibiotics tested.

Figure 2
Antimicrobial resistance (AMR) of isolated bacterial strains using the Kirby-Bauer disk diffusion method.

The highest resistance was observed against cefixime (90%), followed by amoxicillin (70%) and ceftriaxone (60%). In contrast, the greatest susceptibility was recorded for levofloxacin (75%), ciprofloxacin (65%), azithromycin (65%), and tetracycline (60%) (Table 4).

Table 4
Antimicrobial resistance (AMR) measurement (mm) of the tested strains against eight antibiotics according to CLSI, 2020 and multidrug resistance (MDR).

Strain ID	Azithromycin (AZM) 15 µg	Ciprofloxacin (CIP) 5 µg	Levofloxacin (LEV) 5 µg	Cefixime (CFM) 5 µg	Ceftriaxone (CRO) 30 µg	Amoxicillin (AML) 10 µg	Tetracycline (TE)30 µg	Cefaclor (CEC) 30 µg	Number of Resistant classes	MDR
G1CF	0 (R)	30 (S)	15 (R)	0 (R)	16 (R)	9 (R)	20 (S)	14 (R)	3	Yes
G1YR	30 (S)	22 (I)	26 (S)	0 (R)	15 (R)	9 (R)	15 (S)	0 (R)	2	No
G2RT	13 (S)	23 (I)	24 (S)	0 (R)	10 (R)	11 (R)	21 (S)	28 (S)	2	No
G2CR	20 (S)	28 (S)	22 (S)	0 (R)	25 (S)	0 (R)	14 (I)	0 (R)	2	No
G2WR	21 (S)	28 (S)	30 (S)	23 (S)	25 (S)	27 (S)	15 (S)	35 (S)	0	No
G4OC	15 (S)	25 (I)	20 (I)	0 (R)	23 (S)	0 (R)	12 (I)	0 (R)	2	No
G4CF	21 (S)	30 (S)	27 (S)	10 (R)	20 (I)	20 (S)	31(S)	30 (S)	1	No
G4WC	0 (R)	30 (S)	20 (I)	0 (R)	15 (R)	0 (R)	15 (S)	0 (R)	3	Yes
G5YCF	14 (S)	35 (S)	26 (S)	0 (R)	21 (I)	0 (R)	13 (I)	0 (R)	2	No
G5YRW	0 (R)	30 (S)	24 (S)	0 (R)	0 (R)	0 (R)	11 (R)	0 (R)	4	Yes
G5WLI	22 (S)	31 (S)	34 (S)	0 (R)	0 (R)	17 (S)	10 (R)	0 (R)	2	No
G6CR	20 (S)	27 (S)	25 (S)	25 (S)	25 (S)	0 (R)	20 (S)	33 (S)	1	No
G6PYF	20 (S)	28 (S)	28 (S)	0 (R)	15 (R)	0 (R)	22 (S)	20 (S)	2	No
S1WF	0 (R)	20 (R)	19 (I)	0 (R)	21 (I)	0 (R)	9 (R)	0 (R)	3	Yes
S1CF	20 (S)	32 (S)	20 (I)	0 (R)	23(S)	0 (R)	11(R)	0 (R)	3	Yes
S2WF	7 (R)	22 (I)	38 (S)	0 (R)	2 (R)	8 (R)	26 (S)	4 (R)	3	Yes
S2CC	35 (S)	38 (S)	29 (S)	0 (R)	13 (R)	21 (S)	28 (S)	30 (S)	1	No
S2CF	23 (I)	24 (I)	32 (S)	0 (R)	16 (R)	22 (S)	30 (S)	34 (S)	2	No
S3WR	0 (R)	30 (S)	30(S)	0 (R)	13 (R)	0 (R)	11(R)	25(S)	4	Yes
S4WIC	20 (S)	25 (I)	24 (S)	0 (R)	15 (R)	20 (S)	22 (S)	30 (S)	1	No

• Periplaneta americana: G1CF, G1YR, G2RT, G2CR, S1WF, S1CF, S2WF, S2CC; Blatta lateralis: G4OC, G4CF, G4WC; Blattella germanica: S3WR, S4WIC, G5YCF, G5YRW, G5WLI; Pycnoscelus surinamensis: G6CR, G6PYF

Phylogenetic analysis

Molecular identification using 16S rRNA gene sequencing with 1,000 bootstrap replicates revealed close homology between the isolated strains and 20 distinct bacterial species (Table 5). Of the total isolates, 13 originated from the gut and seven from the external surface of freshly collected cockroach specimens. Gut-associated strains showed close genetic similarity to a range of clinically relevant bacteria, including Staphylococcus aureus, Stenotrophomonas maltophilia, Bacillus paramycoides, Escherichia coli, Pseudomonas aeruginosa, Klebsiella quasipneumoniae, Bacillus subtilis, Klebsiella pneumoniae, Cronobacter sakazakii, Paenibacillus thiaminolyticus, Paenibacillus dendritiformis, Klebsiella variicola, and Citrobacter freundii. In contrast, surface-associated isolates showed the highest sequence similarity to species such as B. atrophaeus, B. safensis, S. haemolyticus, S. gallinarum, B. pumilus, B. cereus, and B. paranthracis (Figure 3). An evolutionary tree was constructed for all 20 bacterial strains using the Neighbor-Joining method, with bootstrap values (e.g., 52%, 54%, 65%, 79%) indicating statistical support at branch nodes, higher values reflecting greater confidence. A major bifurcation in the tree, supported by a bootstrap value of 79%, revealed two primary clades stemming from a common ancestor. Notably, both gut- and surface-derived isolates were distributed across these clades, indicating no strict phylogenetic separation between internal and external microbiota. For instance, strains G6CR, S3WR, G4WC, G6PYF, G5YCF, G1YR, G1CF, G2CR, and G2WR clustered into a large clade, suggesting a shared evolutionary lineage. The second major clade included S2WF, S2CF, G2RT, G4CF, S4WIC, S1WF, S2CC, and S1CF. Interestingly, three gut isolates (G4OC, G5WLI, and G5YRW) formed a distinct minor clade at the end of the tree, suggesting a unique evolutionary trajectory (Figure 4).

Table 5
Closely related bacterial species identified by the Maximum Likelihood Method (MEGA X) for each strain.

Strain ID	Cockroach species	Biotypes	Closest bacterial species
G1CF	Periplaneta americana	Hospital, Gujranwala City	Staphylococcus aureus
G1YR	Periplaneta americana	Canteen, Wazirabad	Stenotrophomonas maltophilia
G2RT	Periplaneta americana	Households, Kamonke	Bacillus paramycoides
G2CR	Periplaneta americana	Households, Gujranwala Saddar	Escherichia coli
G2WR	Periplaneta americana	Hospital, Gujranwala City	Pseudomonas aeruginosa
G4OC	Blatta lateralis	Households, Nowshera Virkan	Klebsiella quasipneumoniae
G4CF	Blatta lateralis	Canteen, Gujranwala city	Bacillus subtilis
G4WC	Blatta lateralis	Households, Gujranwala Saddar	Klebsiella pneumoniae
G5YCF	Blattella germanica	Hospital, Kamonke	Cronobacter sakazakii
G5YRW	Blattella germanica	Households, Wazirabad	Paenibacillus thiaminolyticus
G5WLI	Blattella germanica	Canteen, Gujranwala Saddar	Paenibacillus dendritiformis
G6CR	Pycnoscelus surinamensis	Households, Wazirabad	Klebsiella variicola
G6PYF	Pycnoscelus surinamensis	Households, Wazirabad	Citrobacter freundii
S1WF	Periplaneta americana	Hospital, Gujranwala City	Bacillus atrophaeus
S1CF	Periplaneta americana	Canteen, Wazirabad	Bacillus safensis
S2WF	Periplaneta americana	Households, Kamonke	Staphylococcus haemolyticus
S2CC	Periplaneta americana	Households, Gujranwala Saddar	Bacillus pumilus
S2CF	Periplaneta americana	Hospital, Gujranwala City	Staphylococcus gallinarum
S3WR	Blattella germanica	Hospital, Kamonke	Bacillus cereus
S4WIC	Blattella germanica	Canteen, Gujranwala Saddar	Bacillus paranthracis

• G = Gut isolate, S = Surface isolate; The number (1–6) represents the individual cockroach from which the strain was isolated. The final letters represent colony morphology as observed on agar plates: CF = Creamy Filamentous, YR = Yellow Round, WR = Whitish Round, WC = White Curled, PYF = Pale Yellow Filamentous, WF = White Filamentous, YF = Yellowish Filamentous, CC = Creamy Curled, CRT = Creamy Round Threadlike, WIC = White Irregular Curled, RT = Round Transparent, OC = Off-white Curled, CR = Creamy Round, YCF = Yellowish Creamy Filamentous, WLI = White Large Irregular, YRW = Yellowish Round Whitish

Figure 3
Phylogenetic relationship among tested bacterial strains based on 16S rRNA gene sequence through the Neighbor Joining method using 1000 bootstrap replicates.

Figure 4
Venn diagram illustrating the distribution of closely related bacterial species isolated from the gut and surface of four cockroach species.

Ecological and pathogenic diversity of cockroach-associated bacteria

Bacterial isolates obtained across five sub divisions were grouped into human pathogens, environmental bacteria, opportunistic pathogens, and entomophilic species (Table 6). Periplaneta americana carried the most diverse bacterial community (ten species), which included important human pathogens such as P. aeruginosa, S. aureus, and E. coli, particularly from hospital and household settings. In contrast, B. germanica mainly was associated with environmental and opportunistic bacteria like B. cereus and C. sakazakii. B. lateralis and P. surinamensis yielded fewer isolates, which included some opportunistic and potentially insect-associated bacteria, such as K. variicola and S. gallinarum (Figure 4).

Table 6
Distribution of bacterial isolates by cockroach species, site, and ecologicalpathogenic category.

Subdivisions	Cockroach Species	Human Pathogens	Environmental Bacteria	Opportunistic / Other	Entomophilic
Gujranwala City	P. americana	P. aeruginosa, S. aureus	B. atrophaeus	--	S. gallinarum
	B. lateralis	--	B. subtilis	--	--
Gujranwala Saddar	B. germanica	--	P. dendritiformis	B. paranthracis	--
	B. lateralis	K. pneumoniae	--	--	--
	P. americana	E. coli	B. pumilus	--	--
Kamonke	P. americana	S. haemolyticus	B. paramycoides	--	--
	B. germanica	B. cereus	--	C. sakazakii	--
Nowshera Virkan	B. lateralis	--	--	K. quasipneumoniae	--
Wazirabad	B. germanica	--	P. thiaminolyticus	--	--
	P. americana	S. maltophilia	B. safensis	--	--
	P. surinamensis	--	--	C. freundii	K. variicola

Discussion

This study reports the first record of Pycnoscelus surinamensis in Pakistan, expanding its known distribution and contributing to the growing documentation of urban cockroach fauna in tropical and subtropical regions. The detection of four cockroach species across diverse habitats demonstrates their ecological adaptability and capacity to act as mechanical and/or biological vectors of pathogens. The widespread occurrence of B. germanica across all habitats and seasons underscores its high adaptation to human-associated environments, consistent with previous findings (Latorre et al., 2022). Its high reproductive rate, ecological plasticity, and resilient gut microbiome, enabling survival in urban settings, likely explain its co-dominance with P. americana (Wang et al., 2000). The association of all four species with at least one bacterial pathogen reinforces their potential role in disease transmission, in line with earlier reports from residential, healthcare, and food-handling environments (Correia et al., 2018; Gaire and Romero, 2020; Mehainaoui et al., 2021). These findings confirm the growing belief that urban cockroaches act as both mechanical and biological carriers of pathogens (Tatfeng et al., 2005).

Biochemical characterization using catalase, citrate, and indole tests generally aligned with expected metabolic profiles of the respective genera. For example, indole-positive reactions in E. coli, Klebsiella spp., and C. freundii are well-documented, as is citrate utilization in Pseudomonas, Paenibacillus, and Bacillus spp. (Fang et al., 2025). Unexpected catalase-negative reactions in B. paramycoides and S. aureus may reflect strain-level variation or environmental modulation of gene expression (Burford-Gorst and Kidd, 2024). While useful for preliminary screening, phenotypic assays require molecular confirmation, particularly for environmental isolates with variable traits (Church et al., 2020).

Molecular identification via 16S rRNA sequencing revealed S. aureus, P. aeruginosa, K. pneumoniae, and E. coli, pathogens implicated in both hospital- and community-acquired infections due to their virulence and antibiotic resistance (Hailemariam et al., 2021). Their presence in densely populated, unsanitary urban environments highlights significant public health concerns. Notably, P. americana harbored the most diverse and resistant strains, supporting prior observations of its importance as a carrier of antibiotic-resistant bacteria (Pai et al., 2005; Islam et al., 2016). The isolation of K. quasipneumoniae, K. pneumoniae, B. subtilis, and E. coli in the guts of B. lateralis and P. americana confirms earlier reports that cockroach guts harbor members of the Firmicutes and Bacteroidetes phyla, often linked to fecal contamination and gastrointestinal infections (Shiota et al., 2010; Makvana and Krilov, 2015).

The isolation of P. aeruginosa from P. americana and C. sakazakii from B. germanica underscores the heightened risk of cockroach infestations in healthcare settings (Neuhauser et al., 2003; Jain et al., 2021; Adeyemi et al., 2022). Other opportunistic pathogens, such as P. thiaminolyticus and P. dendritiformis, further exemplify the microbial diversity in cockroach microbiota that may affect vulnerable populations.

Interestingly, P. surinamensis, though less abundant, contained Klebsiella variicola and C. freundii, both known to cause respiratory, urinary, and intestinal infections (Wang et al., 2000; Garza-Ramos et al., 2018). Environmental isolates such as Bacillus atrophaeus, B. safensis, and B. pumilus were recovered from P. americana, and B. cereus and B. paranthracis from B. germanica, indicating exposure to soil- or dust-borne bacteria that can be pathogenic or toxin-producing (Benardini et al., 2003; Malik et al., 2013; Chu et al., 2019). Of particular concern is B. cereus, a known foodborne pathogen with notable resistance traits and stress tolerance (Bottone, 2010; Isaac et al., 2014; Abdolmaleki et al., 2019).

Antimicrobial susceptibility testing revealed widespread β-lactam resistance, particularly to cephalosporins (cefixime, ceftriaxone, cefaclor) and amoxicillin, consistent with global trends linked to overuse in human and veterinary medicine (Alraey et al., 2025). Tetracycline resistance, although moderate, is concerning due to its frequent linkage to mobile genetic elements that facilitate horizontal gene transfer (Grossman, 2016). The high proportion of isolates (85%) with a MAR index > 0.2 suggests origin from environments under strong antibiotic pressure such as hospitals, canteens, and wastewater-contaminated areas (Krumperman, 1983; Muwonge et al., 2025).

Widespread β-lactam resistance (e.g., cefixime, amoxicillin) aligns with global trends, while relatively higher susceptibility to fluoroquinolones and tetracyclines may reflect limited local use. While spontaneous mutations can confer resistance, horizontal gene transfer (HGT) is a faster, more influential driver of resistance dissemination in microbial communities.

Overall, these findings demonstrate strong links between cockroach behavior, habitat type, and microbial acquisition, with gut microbiota likely reflecting long-term colonization and surface microbiota indicating recent environmental contact. The potential for HGT amplifies the risk of pathogen spread, particularly in overcrowded, unsanitary urban settings. Integrating cockroach surveillance into vector control and antimicrobial resistance monitoring programs, especially in healthcare and food-handling environments, should be prioritized to mitigate public health risks.

Conclusion

This study identified four cockroach species across five sub divisions of District Gujranwala, with B. germanica emerging as the most abundant and P. surinamensis reported for the first time in Pakistan. Overall, bacterial isolates exhibited high resistance to cephalosporins and penicillins, while fluoroquinolones showed the lowest resistance rates. The notable prevalence of multidrug-resistant (MDR) strains (35%), most with a Multiple Antibiotic Resistance (MAR) index greater than 0.2, underscores the significant public health risk posed by cockroaches as reservoirs of MDR bacteria. Phylogenetic analysis revealed that twenty strains from the four cockroach species were closely related to pathogenic bacteria, excepti B. pumilus, which is generally considered non-pathogenic or beneficial. Given the higher infestation rates in human dwellings and the associated health hazards, implementation of Integrated Pest Management (IPM) strategies is strongly recommended to reduce cockroach populations and curb the dissemination of pathogenic and MDR bacteria in urban environments.

• Funding
  There was no funding available for this research project.
• Research permit
  This study was approved by the Ethical Committee of the University of Gujrat.
• Compliance with ethical standards
  All efforts were taken to minimize pain and discomfort to the animal while conducting this research according to the ethical protocol of the University of Gujrat.

References

• Abdolmaleki, Z., Mashak, Z., Safarpoor Dehkordi, F., 2019. Phenotypic and genotypic characterization of antibiotic resistance in the methicillin-resistant Staphylococcus aureus strains isolated from hospital cockroaches. Antimicrob. Resist. Infect. Control 8 (1), 54. http://doi.org/10.1186/s13756-019-0505-7
  » http://doi.org/10.1186/s13756-019-0505-7
• Adedara, I. A., Mohammed, K. A., Da-Silva, O. F., Salaudeen, F. A., Gonçalves, F. L., Rosemberg, D. B., Aschner, M., Rocha, J. B., Farombi, E. O., 2022. Utility of cockroach as a model organism in the assessment of toxicological impacts of environmental pollutants. Environ. Adv. 8, 100195. http://doi.org/10.1016/j.envadv.2022.100195
  » http://doi.org/10.1016/j.envadv.2022.100195
• Adeyemi, F. M., Ojo, O. O., Badejo, A. A., Oyedara, O. O., Olaitan, J. O., Adetunji, C. O., Hefft, D. I., Ogunjobi, A. A., Akinde, S. B., 2022. Integrated poultry-fish farming system encourages multidrug-resistant gram-negative bacteria dissemination in pond environment and fishes. Aquaculture 548, 737558. http://doi.org/10.1016/j.aquaculture.2021.737558
  » http://doi.org/10.1016/j.aquaculture.2021.737558
• Alraey, Y., Assiry, M. M., Ahmad, I., Alqahtani, A., Basheer, N., AlAsiri, M. A., Alshehri, S. A., Alhamhhum, S. M., Alhefdi, S. M., Khan, M. S., Saleem, M., Bawazeer, A. A. S., 2025. Antimicrobial resistance and beta-lactamase gene distribution among clinical isolates: a two-year cohort study. Sci. Rep. 15 (1), 23951. http://doi.org/10.1038/s41598-025-08445-w
  » http://doi.org/10.1038/s41598-025-08445-w
• Baig, M. B., Ehrenreich, J. H., 1997. The potential prospects and problems of agroforestry in Pakistan-1: research and development. Pak. J. Agric. Sci. 34, 22-28.
• Benardini, J. N., Sawyer, J., Venkateswaran, K., Nicholson, W. L., 2003. Spore UV and acceleration resistance of endolithic Bacillus pumilus and Bacillus subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia. Astrobiology 3 (4), 709-717. http://doi.org/10.1089/153110703322736033
  » http://doi.org/10.1089/153110703322736033
• Bilal, H., Khan, M. N., Rehman, T., Hameed, M. F., Yang, X., 2021. Antibiotic resistance in Pakistan: a systematic review of past decade. BMC Infect. Dis. 21 (1), 244. http://doi.org/10.1186/s12879-021-05906-1
  » http://doi.org/10.1186/s12879-021-05906-1
• Bottone, E. J., 2010. Bacillus cereus, a volatile human pathogen. Clin. Microbiol. Rev. 23 (2), 382-398. http://doi.org/10.1128/CMR.00073-09
  » http://doi.org/10.1128/CMR.00073-09
• Bouamamaa, L., Sorlozano, A., Laglaoui, A., Lebbadi, M., Aarab, A., Gutierrez, J., 2010. Antibiotic resistance patterns of bacterial strains isolated from Periplaneta americana and Musca domestica in Tangier, Morocco. J. Infect. Dev. Ctries. 4 (04), 194-201. http://doi.org/10.3855/jidc.336
  » http://doi.org/10.3855/jidc.336
• Burford-Gorst, C. M., Kidd, S. P., 2024. Phenotypic variation in Staphylococcus aureus during colonisation involves antibiotic-tolerant cell types. Antibiotics 13 (9), 845. http://doi.org/10.3390/antibiotics13090845
  » http://doi.org/10.3390/antibiotics13090845
• Choate, P., Burns, S., Olsen, L., Richman, D., Perez, O., Patnaude, M., Pluke, R., 2008. A dichotomous key for the identification of the cockroach fauna (Insecta: Blattaria) of Florida. Florida Entomologist 72 (4), 612-617.
• Chompoosri, J., Thavara, U., Tawatsin, A., Sathantriphop, S., Yi, T., 2004. Cockroach surveys in the northern region of Thailand and guangxi province of China. Southeast Asian J. Trop. Med. Public Health 35 (Suppl. 2), 46-49.
• Christopher, Y., Wcislo, W. T., Martínez‐Luis, S., Hughes, W. O., Gerardo, N. M., Fernández‐Marín, H., 2021. Disease management in two sympatric Apterostigma fungus‐growing ants for controlling the parasitic fungus Escovopsis. Ecol. Evol. 11 (11), 6041-6052. http://doi.org/10.1002/ece3.7379
  » http://doi.org/10.1002/ece3.7379
• Chu, J., Wang, Y., Zhao, B., Zhang, X., Liu, K., Mao, L., Kalamiyets, E., 2019. Isolation and identification of new antibacterial compounds from Bacillus pumilus. Appl. Microbiol. Biotechnol. 103 (20), 8375-8381. http://doi.org/10.1007/s00253-019-10083-y
  » http://doi.org/10.1007/s00253-019-10083-y
• Church, D. L., Cerutti, L., Gürtler, A., Griener, T., Zelazny, A., Emler, S., 2020. Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clin. Microbiol. Rev. 33 (4), e00053-19. http://doi.org/10.1128/CMR.00053-19
  » http://doi.org/10.1128/CMR.00053-19
• CLSI. 2020. Performance Standards for Antimicrobial Susceptibility Testing, 30th ed. Clinical and Laboratory Standards Institute, Wayne, PA.
• Correia, D. S., Passos, S. R., Proenca, D. N., Morais, P. V., Xavier, G. R., Correia, M. E. F., 2018. Microbial diversity associated to the intestinal tract of soil invertebrates. Appl. Soil Ecol. 131, 38-46. http://doi.org/10.1016/j.apsoil.2018.07.009
  » http://doi.org/10.1016/j.apsoil.2018.07.009
• Dallarés, S., Padrós, F., Cartes, J. E., Solé, M., Carrassón, M., 2017. The parasite community of the sharks Galeus melastomus, Etmopterus spinax and Centroscymnus coelolepis from the NW Mediterranean deep-sea in relation to feeding ecology and health condition of the host and environmental gradients and variables. Deep Sea Res. Part I Oceanogr. Res. Pap. 129, 41-58. http://doi.org/10.1016/j.dsr.2017.09.007
  » http://doi.org/10.1016/j.dsr.2017.09.007
• Faheem, M., Mati, N., Matiullah, 2007. Seasonal variation in indoor radon concentrations in dwellings in six districts of the Punjab province, Pakistan. J. Radiol. Prot. 27 (4), 493-500. http://doi.org/10.1088/0952-4746/27/4/N01
  » http://doi.org/10.1088/0952-4746/27/4/N01
• Fang, X., Chen, Y., Hu, L., Gu, S., Zhu, J., Hang, Y., Cao, X., Xiao, Y., Luo, H., Zhao, C., Xiao, L., Zhong, Q., 2025. Modulation of virulence and metabolic profiles in Klebsiella pneumoniae under indole-mediated stress response. Front. Cell. Infect. Microbiol. 15, 1546991. http://doi.org/10.3389/fcimb.2025.1546991
  » http://doi.org/10.3389/fcimb.2025.1546991
• Fischer, O., Matlova, L., Dvorska, L., Svastova, P., Pavlik, I., 2003. Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis. Med. Vet. Entomol. 17 (2), 145-150. http://doi.org/10.1046/j.1365-2915.2003.00417.x
  » http://doi.org/10.1046/j.1365-2915.2003.00417.x
• Gaire, S., Romero, A., 2020. Comparative efficacy of residual insecticides against the Turkestan cockroach, Blatta lateralis,(Blattodea: Blattidae) on different substrates. Insects 11 (8), 477. http://doi.org/10.3390/insects11080477
  » http://doi.org/10.3390/insects11080477
• Garrity, G. M., Holt, J. G., 2001. The road map to the manual. Bergey’s Man. Syst. Bacteriol. 1, 119-169.
• Garza-Ramos, U., Barrios-Camacho, H., Moreno-Domínguez, S., Toribio-Jiménez, J., Jardón-Pineda, D., Cuevas-Peña, J., Sánchez-Pérez, A., Duran-Bedolla, J., Olguín-Rodriguez, J., Román-Román, A., 2018. Phenotypic and molecular characterization of Klebsiella spp. isolates causing community-acquired infections. New Microbes New Infect. 23, 17-27. http://doi.org/10.1016/j.nmni.2018.02.002
  » http://doi.org/10.1016/j.nmni.2018.02.002
• Ghasemi-Dehkordi, P., Doosti, A., Doosti, E., Noshadi, E., Arshi, A., 2016. Antimicrobial susceptibility patterns of Escherichia coli isolates from cockroaches in southwestern Iran. Bulg. J. Vet. Med. 19 (1), 63-71. http://doi.org/10.15547/bjvm.860
  » http://doi.org/10.15547/bjvm.860
• Graczyk, T. K., Knight, R., Tamang, L., 2005. Mechanical transmission of human protozoan parasites by insects. Clin. Microbiol. Rev. 18 (1), 128-132. http://doi.org/10.1128/CMR.18.1.128-132.2005
  » http://doi.org/10.1128/CMR.18.1.128-132.2005
• Grossman, T. H., 2016. Tetracycline antibiotics and resistance. Cold Spring Harb. Perspect. Med. 6 (4), a025387. http://doi.org/10.1101/cshperspect.a025387
  » http://doi.org/10.1101/cshperspect.a025387
• Guzman, J., Vilcinskas, A., 2020. Bacteria associated with cockroaches: health risk or biotechnological opportunity? Appl. Microbiol. Biotechnol. 104 (24), 10369-10387. http://doi.org/10.1007/s00253-020-10973-6
  » http://doi.org/10.1007/s00253-020-10973-6
• Hagenbuch, B. E., Koehler, P. G., Patterson, R. S., Brenner, R. J., 1988. Peridomestic cockroaches (Orthoptera: Blattidae) of Florida: their species composition and suppression. J. Med. Entomol. 25 (5), 377-380. http://doi.org/10.1093/jmedent/25.5.377
  » http://doi.org/10.1093/jmedent/25.5.377
• Hailemariam, M., Alemayehu, T., Tadesse, B., Nigussie, N., Agegnehu, A., Habtemariam, T., Ali, M., Mitiku, E., Azerefegne, E., 2021. Major bacterial isolate and antibiotic resistance from routine clinical samples in Southern Ethiopia. Sci. Rep. 11 (1), 19710. http://doi.org/10.1038/s41598-021-99272-2
  » http://doi.org/10.1038/s41598-021-99272-2
• Isaac, C., Orue, P. O., Iyamu, M. I., Ehiaghe, J. I., Isaac, O., 2014. Comparative analysis of pathogenic organisms in cockroaches from different community settings in Edo State, Nigeria. Korean J. Parasitol. 52 (2), 177-181. http://doi.org/10.3347/kjp.2014.52.2.177
  » http://doi.org/10.3347/kjp.2014.52.2.177
• Islam, A., Nath, A. D., Islam, K., Islam, S., Chakma, S., Hossain, M. B., Al-Faruq, A., Hassan, M. M., 2016. Isolation, identification and antimicrobial resistance profile of Staphylococcus aureus in Cockroaches (Periplaneta americana). J. Adv. Vet. Anim. Res. 3 (3), 221-228. http://doi.org/10.5455/javar.2016.c153
  » http://doi.org/10.5455/javar.2016.c153
• Jacobs, L., Chenia, H. Y., 2007. Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int. J. Food Microbiol. 114 (3), 295-306. http://doi.org/10.1016/j.ijfoodmicro.2006.09.030
  » http://doi.org/10.1016/j.ijfoodmicro.2006.09.030
• Jain, N., Jansone, I., Obidenova, T., Simanis, R., Meisters, J., Straupmane, D., Reinis, A., 2021. Antimicrobial resistance in nosocomial isolates of gram-negative bacteria: public health implications in the latvian context. Antibiotics 10 (7), 791. http://doi.org/10.3390/antibiotics10070791
  » http://doi.org/10.3390/antibiotics10070791
• Jalil, N., Keyhani, A., Hasan, M.-K. S., Mahdi, M., Monireh, M., Atefeh, B., 2012. Cockroaches’ bacterial infections in wards of hospitals, Hamedan city, west of Iran. Asian Pac. J. Trop. Dis. 2 (5), 381-384. http://doi.org/10.1016/S2222-1808(12)60083-8
  » http://doi.org/10.1016/S2222-1808(12)60083-8
• Javaid, A., Hussain, M., Aftab, K., Malik, M., Umar, M., Iqbal, T., 2021. Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding. Braz. J. Biol. 84, e249664. http://doi.org/10.1590/1519-6984.249664
  » http://doi.org/10.1590/1519-6984.249664
• Kabantiyok, D., Gyang, M. D., Agada, G. O., Ogundeji, A., Nyam, D., Uhiara, U. G., Abiayi, E., Dashe, Y., Ngulukun, S., Muhammad, M., Adegboye, O. A., Emeto, T. I., 2023. Analysis of retrospective Laboratory data on the burden of bacterial pathogens isolated at the National Veterinary Research Institute Nigeria, 2018–2021. Vet. Sci. 10 (8), 505. http://doi.org/10.3390/vetsci10080505
  » http://doi.org/10.3390/vetsci10080505
• Kamran, K., Schapheer, C., Ali, A., Kamran Taj, M., Siddiqui, S., Maldonado, A. K., Iqbal, A., Arif, S., Villagra, C., 2021. Spatial distribution of synanthropic cockroaches found in Quetta, Pakistan and antibiotic‐resistant bacteria strains found in Shelfordella lateralis (Walker, 1868)(Blattodea: blattidae). Entomol. Res. 51 (12), 624-638. http://doi.org/10.1111/1748-5967.12556
  » http://doi.org/10.1111/1748-5967.12556
• Khan, A., Tauseef, I., Aalia, B., Khan, M. A., Akbar, S., Sultana, N., Haleem, K. S., 2018. Age-related variations in the in vitro bactericidal activity of human sera against Pseudomonas aeruginosa. Cent. Eur. J. Immunol. 43 (1), 18-25. http://doi.org/10.5114/ceji.2018.74869
  » http://doi.org/10.5114/ceji.2018.74869
• Krumperman, P. H., 1983. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl. Environ. Microbiol. 46 (1), 165-170. http://doi.org/10.1128/aem.46.1.165-170.1983
  » http://doi.org/10.1128/aem.46.1.165-170.1983
• Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35 (6), 1547-1549. http://doi.org/10.1093/molbev/msy096
  » http://doi.org/10.1093/molbev/msy096
• Kundera, I. N., Sapu, E. H., Bialangi, M., 2020. Identification of bacteria on Cockroach feet (Periplaneta americana) in resident Bay of Palu permai and sensitivity test against antibiotics. Techno. J. Penelitian 9 (1), 353-362. https://doi.org/10.33387/tjp.v9i1.1758.
• Latorre, A., Domínguez-Santos, R., García-Ferris, C., Gil, R., 2022. Of cockroaches and symbionts: recent advances in the characterization of the relationship between Blattella germanica and its dual symbiotic system. Life 12 (2), 290. http://doi.org/10.3390/life12020290
  » http://doi.org/10.3390/life12020290
• Liu, A., Chen, S., Wang, M., Liu, D., Chang, R., Wang, Z., Lin, X., Bai, B., Ahammed, G. J., 2016. Arbuscular mycorrhizal fungus alleviates chilling stress by boosting redox poise and antioxidant potential of tomato seedlings. J. Plant Growth Regul. 35 (1), 109-120. http://doi.org/10.1007/s00344-015-9511-z
  » http://doi.org/10.1007/s00344-015-9511-z
• Máca, O., Kouba, M., Korpimäki, E., González-Solís, D., 2021. Molecular identification of Sarcocystis sp.(Apicomplexa, Sarcocystidae) in offspring of Tengmalm’s owls, Aegolius funereus (Aves, Strigidae). Front. Vet. Sci. 8, 804096. http://doi.org/10.3389/fvets.2021.804096
  » http://doi.org/10.3389/fvets.2021.804096
• Magiorakos, A.-P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M., Giske, C., Harbarth, S., Hindler, J., Kahlmeter, G., Olsson-Liljequist, B., 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18 (3), 268-281.
• Mahmood, A., Mahmood, A., Malik, R. N., Shinwari, Z. K., 2013. Indigenous knowledge of medicinal plants from Gujranwala district, Pakistan. J. Ethnopharmacol. 148 (2), 714-723. http://doi.org/10.1016/j.jep.2013.05.035
  » http://doi.org/10.1016/j.jep.2013.05.035
• Makvana, S., Krilov, L. R., 2015. Escherichia coli infections. Pediatrics 36 (4), 167-171. https://doi.org/10.1542/pir.36-4-167.
• Malik, K., Jamil, A., Arshad, A., 2013. Study of pathogenic microorganisms in the external body parts of American cockroach (Periplaneta americana) collected from different kitchens. IOSR J. Pharm. Biol. Sci. 7 (6), 45-48. http://doi.org/10.9790/3008-0764548
  » http://doi.org/10.9790/3008-0764548
• Malinga, N. Z., Shobo, C. O., Molechan, C., Amoako, D. G., Zishiri, O. T., Bester, L. A., 2022. Molecular surveillance and dissemination of Klebsiella pneumoniae on frequently encountered surfaces in South African Public Hospitals. Microb. Drug Resist. 28 (3), 306-316. https://doi.org/10.1089/mdr.2020.0546.
• Mehainaoui, A., Menasria, T., Benouagueni, S., Benhadj, M., Lalaoui, R., Gacemi‐Kirane, D., 2021. Rapid screening and characterization of bacteria associated with hospital cockroaches (Blattella germanica L.) using MALDI‐TOF mass spectrometry. J. Appl. Microbiol. 130 (3), 960-970. http://doi.org/10.1111/jam.14803
  » http://doi.org/10.1111/jam.14803
• Memona, H., Manzoor, F., Riaz, S., 2017. Species diversity and distributional pattern of cockroaches in Lahore, Pakistan. J. Arthropod Borne Dis. 11 (2), 249-259.
• Mpuchane, S., Allotey, J., Matsheka, I., Simpanya, M., Coetzee, S., Jordaan, A., Mrema, N., Gashe, B., 2006. Carriage of micro-organisms by domestic cockroaches and implications on food safety. Int. J. Trop. Insect Sci. 26 (3), 166-175. https://doi.org/10.1079/IJT20063141.
• Mullins, D. E., 2015. Physiology of environmental adaptations and resource acquisition in cockroaches. Annu. Rev. Entomol. 60 (1), 473-492. http://doi.org/10.1146/annurev-ento-011613-162036
  » http://doi.org/10.1146/annurev-ento-011613-162036
• Muwonge, K. M., Ndagire, H., Mulindwa, J., Twesigye, C. K., 2025. Multiple antimicrobial resistance indices of Staphylococcus aureus from the nares of goats and slaughterhouse attendants in Kampala city, Uganda–a cross sectional study. BMC Microbiol. 25 (1), 162. http://doi.org/10.1186/s12866-025-03891-y
  » http://doi.org/10.1186/s12866-025-03891-y
• Naher, A., Afroz, S., Hamid, S., 2018. Cockroach associated foodborne pathogens: distribution and antibiogram. Bangladesh Med. Res. Counc. Bull. 44 (1), 30-38. http://doi.org/10.3329/bmrcb.v44i1.36802
  » http://doi.org/10.3329/bmrcb.v44i1.36802
• Nasirian, H., 2017. Infestation of cockroaches (Insecta: Blattaria) in the human dwelling environments: a systematic review and meta-analysis. Acta Trop. 167, 86-98. http://doi.org/10.1016/j.actatropica.2016.12.019
  » http://doi.org/10.1016/j.actatropica.2016.12.019
• Nasirian, H., 2019. Contamination of cockroaches (Insecta: Blattaria) by medically important bacteriae: a systematic review and meta-analysis. J. Med. Entomol. 56 (6), 1534-1554. http://doi.org/10.1093/jme/tjz095
  » http://doi.org/10.1093/jme/tjz095
• Neuhauser, M. M., Weinstein, R. A., Rydman, R., Danziger, L. H., Karam, G., Quinn, J. P., 2003. Antibiotic resistance among gram-negative bacilli in US intensive care units: implications for fluoroquinolone use. JAMA 289 (7), 885-888. http://doi.org/10.1001/jama.289.7.885
  » http://doi.org/10.1001/jama.289.7.885
• Ofoedu, C. E., Iwouno, J. O., Agunwah, I. M., Obodoechi, P. Z., Okpala, C. O. R., Korzeniowska, M., 2021. Bacterial contamination of Nigerian currency notes: A comparative analysis of different denominations recovered from local food vendors. PeerJ 9, e10795. http://doi.org/10.7717/peerj.10795
  » http://doi.org/10.7717/peerj.10795
• Pai, H.-H., Chen, W.-C., Peng, C.-F., 2005. Isolation of bacteria with antibiotic resistance from household cockroaches (Periplaneta americana and Blattella germanica). Acta Trop. 93 (3), 259-265. http://doi.org/10.1016/j.actatropica.2004.11.006
  » http://doi.org/10.1016/j.actatropica.2004.11.006
• Patel, S., Meher, B., 2016. A review on emerging frontiers of house dust mite and cockroach allergy research. Allergol. Immunopathol. 44 (6), 580-593. http://doi.org/10.1016/j.aller.2015.11.001
  » http://doi.org/10.1016/j.aller.2015.11.001
• Peterson, R. K., Shurdut, B. A., 1999. Human health risks from cockroaches and cockroach management: a risk analysis approach. Am. Entomol. 45 (3), 142-148. http://doi.org/10.1093/ae/45.3.142
  » http://doi.org/10.1093/ae/45.3.142
• Sharawi, S. E., 2021. Overview on the possible role of transmission COVID-19 by cockroaches (Blattodea: blattidae). Internation J. Entomol. Res. 6 (4), 163-166. https://doi.org/10.17582/journal.aavs/2023/11.6.928.932.
• Shiota, M., Saitou, K., Mizumoto, H., Matsusaka, M., Agata, N., Nakayama, M., Kage, M., Tatsumi, S., Okamoto, A., Yamaguchi, S., Ohta, M., Hata, D., 2010. Rapid detoxification of cereulide in Bacillus cereus food poisoning. Pediatrics 125 (4), e951-e955. http://doi.org/10.1542/peds.2009-2319
  » http://doi.org/10.1542/peds.2009-2319
• Solomon, F., Kibru, G., Ali, S., 2018. Multidrug-resistant pattern of food borne illness associated bacteria isolated from cockroaches in meal serving facilities, Jimma, Ethiopia. Afr. Health Sci. 18 (1), 32-40. http://doi.org/10.4314/ahs.v18i1.6
  » http://doi.org/10.4314/ahs.v18i1.6
• Soureshjani, E. H., Doosti, A., 2013. Examination methicillin-resistant Staphylococcus aureus (MRSA) prevalence in cockroaches from hospital in Chaharmahal-va-Bakhtiari province, Iran by polymerase chain reaction (PCR). Int. J. Med. Med. Sci. 5 (7), 324-329. https://doi.org/10.5897/IJMMS2013.0955.
• Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10 (3), 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023.
• Tatfeng, Y., Usuanlele, M., Orukpe, A., Digban, A., Okodua, M., Oviasogie, F., Turay, A., 2005. Mechanical transmission of pathogenic organisms: the role of cockroaches. J. Vector Borne Dis. 42 (4), 129.
• Turner, M., Peta, V., Pietri, J. E., 2022. New insight into the relationship between Salmonella Typhimurium and the German cockroach suggests active mechanisms of vector-borne transmission. Res. Microbiol. 173 (3), 103920. http://doi.org/10.1016/j.resmic.2021.103920
  » http://doi.org/10.1016/j.resmic.2021.103920
• Varaldo, P. E., 2002. Antimicrobial resistance and susceptibility testing: an evergreen topic. J. Antimicrob. Chemother. 50 (1), 1-4. http://doi.org/10.1093/jac/dkf093
  » http://doi.org/10.1093/jac/dkf093
• Wang, C., Bischoff, E., Eiden, A. L., Zha, C., Cooper, R., Graber, J. M., 2019. Residents attitudes and home sanitation predict presence of German cockroaches (Blattodea: Ectobiidae) in apartments for low-income senior residents. J. Econ. Entomol. 112 (1), 284-289. http://doi.org/10.1093/jee/toy307
  » http://doi.org/10.1093/jee/toy307
• Wang, J.-T., Chang, S.-C., Chen, Y.-C., Luh, K.-T., 2000. Comparison of antimicrobial susceptibility of Citrobacter freundii isolates in two different time periods. J. Microbiol. Immunol. Infect. 33 (4), 258-262.
• Weinstein, M. P., Lewis 2nd, J. S., 2020. The clinical and laboratory standards institute subcommittee on antimicrobial susceptibility testing: background, organization, functions, and processes. J. Clin. Microbiol. 58 (3), e01864-19. http://doi.org/10.1128/JCM.01864-19
  » http://doi.org/10.1128/JCM.01864-19
• Weisburg, W. G., Barns, S. M., Pelletier, D. A., Lane, D. J., 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173 (2), 697-703.
• Zarchi, A. A. K., Vatani, H., 2009. A survey on species and prevalence rate of bacterial agents isolated from cockroaches in three hospitals. Vector Borne Zoonotic Dis. 9 (2), 197-200. http://doi.org/10.1089/vbz.2007.0230
  » http://doi.org/10.1089/vbz.2007.0230

Edited by

• Associate Editor:
  Sérvio Pontes Ribeiro

Publication Dates

• Publication in this collection
  07 Nov 2025
• Date of issue
  2025

History

• Received
  25 May 2025
• Accepted
  02 Sept 2025