Isolation and characterization of pathogenic Klebsiella pneumoniae strains from lettuce: a potential source of antibiotic resistance and development of a mathematical model for ANOVA results

Abstract

Introduction: This study aimed to evaluate the prevalence of Klebsiella pneumoniae contamination in raw lettuce from Risalpur, Pakistan, and to analyze the antibiotic susceptibility profiles of the isolated strains. The presence of foodborne pathogens such as K. pneumoniae poses significant public health risks, particularly in regions with suboptimal hygiene practices and improper food handling.

Methods: Lettuce samples were collected from various sources in Risalpur and screened for K. pneumoniae. Antimicrobial susceptibility testing was performed to evaluate the effectiveness of various antibiotics against the isolated strains. Statistical analyses, including ANOVA and linear regression, were conducted to assess differences in inhibition zones and to predict antibiotic effectiveness based on concentration.

Results: The results revealed a significant prevalence of K. pneumoniae in the lettuce samples, highlighting the risks associated with poor hygiene, transportation, storage, and contaminated irrigation water. The isolated strains exhibited high susceptibility to gentamicin but demonstrated notable resistance to doxycycline, vancomycin, and ticarcillin. Multidrug-resistant (MDR) strains were identified. ANOVA showed significant differences in inhibition zones, and the linear regression model predicted a Zone of Inhibition based on antibiotic concentration (β₀ = 10.6667, β₁ = 0.4556).

Discussion: The identification of MDR strains of K. pneumoniae underscores the urgent need for enhanced antibiotic stewardship and food safety protocols to manage foodborne pathogens. Improved hygiene practices throughout the food production and supply chain are critical to mitigate health risks and address the challenge of growing antibiotic resistance.

Keywords: Klebsiella pneumoniae; antibiotic resistance; contamination; food safety; lettuce.

Figure 1

Visualization of antimicrobial susceptibility test findings for the K. pneumoniae strains. Each image shows inhibition zones indicating the effectiveness of different antibiotics.

Figure 2

Bar chart illustrating the inhibition zone diameters for different antibiotics tested against Sample Skp1(a).

Figure 3

ANOVA results for Sample Skp1(a), depicting the analysis of variance across different antibiotics.

Figure 4

Visual representation of the zone of inhibition for various antibiotics tested against Sample Skp1(b).

Figure 5

ANOVA results for Sample Skp1(b), showing the statistical analysis of inhibition zones for different antibiotics.

Figure 6

Zone of inhibition illustrating the various antibiotics against Klebsiella pneumoniae for Sample Skp2(a).

Figure 7

ANOVA results for Sample Skp2(b), showing the statistical analysis of inhibition zone data.

Figure 8

Inhibition zones of antibiotics against Klebsiella pneumoniae for Sample SKp2(b).

Figure 9

Bar plot illustrating the ANOVA results for Sample SKp3(a), showing the variation in inhibition zones among different antibiotics.

Figure 10

Bar plot illustrating the measurement of zone of inhibition for Sample SKp3(a).

Figure 11

Bar plot measuring the zone of inhibition for Sample SKp3(b).

Figure 12

Detailed antibiotic zone of inhibition measurements against Klebsiella pneumoniae for Sample SKp3(b).

Figure 13

Utilization of specific PCR primers for detecting and analyzing qnr genes within K. pneumoniae isolates obtained from raw vegetables.

Figure 14

Results of antibiotic susceptibility testing for K. pneumoniae strains.

Figure 15

Agarose gel electrophoresis of PCR products. The gel, prepared with 1.5% agarose and stained with ethidium bromide, is visualized under UV light. The DNA ladder (L) is annotated with size markers (in base pairs) to the left of the gel. Lane 1: DNA Ladder with size markers; Lane 2: Positive Control (known Klebsiella pneumoniae DNA); Lane 3: Negative Control (PCR reagents without DNA); Lanes 4–6: Samples (identified by separate labels). Heavyweight (HW) bands, representing larger DNA fragments, migrate slower, while lightweight (LW) bands, representing smaller DNA fragments, migrate faster. The gel provides insight into the presence and size of target genes.

Figure 16

Graphical representation of the ANOVA results. The bar plot illustrates the F-values for each sample, while the line plot depicts the corresponding p-values, highlighting the statistical significance of the results.

Figure 17

Bar plot showing the distribution of isolates that are resistant, intermediate, and sensitive to each tested antibiotic. This visualization aids in understanding the overall resistance patterns observed in the Klebsiella pneumoniae isolates.

Figure 18

Bar plot showing the mean differences and line plot illustrating p-values from Tukey's HSD test for pairwise comparisons of antibiotic effectiveness. The bar plot highlights significant differences between antibiotics, while the line plot provides the p-values associated with these differences.

Figure 19

Bar plot illustrating the contamination rates of lettuce samples across different storage durations. The plot shows a clear increase in contamination rates with extended storage time, emphasizing the effect of aging on the contamination levels.

Figure 20

Simulation of antibiotic zone of inhibition based on linear regression. The figure illustrates the predicted relationship between antibiotic types and the Zone of Inhibition, highlighting the regression line and the associated data variability.