Resistotyping of Salmonella spp. and Staphylococcus aureus from Milk and Milk Products Sold in Sabon-Gari and Zaria Local Government Areas of Kaduna State, Nigeria

Mechanisms


Introduction
Milk is the fluid secreted by female mammals for nourishing offspring.It comprises a mixture of complex chemical substances which include fat, protein, lactose, and some mineral matters in the colloidal state in the form of a true solution [1].
Resistotyping involves grouping bacterial isolates based on resistance patterns to a set of randomly chosen antibiotics peculiar to specific strains by phenotypic methods.Antimicrobial resistance (AMR) is on the increase and has posed a major public health concern, severely limiting therapeutic options in clinical settings [2].

Study Area
The study area included two (2) local government areas (Soban-gari and Zaria) in Kaduna state, see Figure 1.

Isolation of organisms
Four hundred (400) samples [100 each] of milk and milk products (Kindirimo, Nono, and Yogurt) sold in Zaria and Sabon-gari.

Pre-Enrichment
Twenty-five (25) grams of the sample were weighed and poured into 225 mL of Buffered peptone water, and incubated at 37 °C for 24 h.

Selective Plating for Staphylococcus aureus
After incubation, a loopful of inoculum (from above) was inoculated on the Mannitol Salt Agar (MSA) plate and incubated at 37 ± 1 °C for 30 h. and observed for growth (yellow halo) indicates Staphylococcus (S.) aureus.

Selective Plating for Salmonella spp.
To isolate Salmonella spp., one (1) ml of pre-enrichment culture was inoculated into Rappaport-Vassiliadis Broth (RV) and incubated at 42 °C for 7 days.After the incubation, it was further cultured on selective agar plates of Salmonella-Shigella Agar (SSA) at 37 °C for 48 h.

Organisms Isolated
A total of 122 isolates; Salmonella spp.(65) and S. aureus (57) were isolated from the samples.

AST Summary
The data presented in Table 1 shows that Salmonella spp.and S. aureus displayed 4 distinct Antimicrobial Resistance Patterns (ARPs) across Groups I, L, O, and P. The largest R population of 44 (36.066%) was in Group O with primary resistance to TET.Then Group P (31.967%) in which all are S. Group I (29.508%)where AMP and TET were R and Group L with the least 02.459%where only AMP was R. From these four (4) groups, representatives were selected for the E-test MIC i.e., 2 per group, total of 16 organisms.Key: Green-Susceptible (≥ 6 mm), Red-Resistance (=5 mm).

E-Test Summary
Table 2 highlights the E-Test pattern.The MIC breakpoints for Salmonella spp., CLSI (AMP S ≤ 08 I16 R ≥ 32; CIP S ≤ 0.25 I0.5 R ≥ 01; GEN S ≤ 02 I04 R ≥ 08, TET S ≤ 04 I08 R ≥ 16).A variation was specifically observed for Salmonella spp., selected isolates from Groups I and L were S to AMP despite being earlier R, the selected organisms that were earlier CIP S were now R and TET resistance was.While for S. aureus, CLSI (AMP S ≤ 04 I-R ≥ 08, CIP S ≤ 01 I02 R ≥ 04, GEN S ≤ 04 I08 R ≥ 16, TET S ≤ 04 I08 R ≥ 16).A variation was also specifically observed for S. aureus, selected isolates from Groups I, and L which were S to AMP are now R, while Group O now showed S when it should be R to only TET.

Discussion
The study conducted by Tamba et al., 2016 [6] on Salmonella isolates showed resistance rates AMP (85.7%),TET (35.7%),CIP (00.0%), and GEN (00.0%).Their findings indicated that AMP is the most resistant drug.Our study shows there is an increase in resistance rates among Salmonella isolates on all the other drugs tested, at TET had (95.385%) on CLSI breakpoint only, CIP (15.385%), and GEN (04.615%) respectively on both CLSI and EUCAST.While AMP showed a drop from 85.7% to 81.538%, TET had an alarming jump from 35.7% to 95.385%.
From Table 1, half of the selected isolates are resistant to AMP and TET, with all being susceptible to CIP AND GEN.However, in Table 2, AMP in Salmonella spp. was 25%R, and S. aureus was 50%R, TET was 37.5%R for both, CIP was 50%R and 25%R respectively, GEN was 00%R and 12.5% R respectively.
In summary, recent research has revealed a surge in antibiotic resistance, particularly in TET, which is the most resistant drug for both organisms.AMP, CIP, and GEN follow in that sequence.Prompt measures must be taken to tackle the escalating issue of antimicrobial resistance (AMR) and curb the proliferation of antibiotic-resistant pathogens.

Table 1 .
AST Summary Based on ZOI.