Multi-drug and biocide-resistant Gram-positive bacteria prevalent on surfaces of veterinary clinics in Benue State, Nigeria

Objective: In this study, the common aerobic Gram-positive bacteria (GPB) contaminating veterinary clinic surfaces were isolated, and their susceptibility to antimicrobial agents (antibiotics and biocides) was determined. Materials and Methods: Standard cultural and biochemical procedures were used to process 62 swab samples collected from different surfaces in 4 veterinary clinics for the isolation and identification of GPB. The agar dilution technique was used to determine the sensitivity of the isolates to disinfectants, while the disc diffusion technique was used to determine the sensitivity to antibiotics. Results: Isolation rates in veterinary clinics I, II, III, and IV were 39.4%, 40.4%, 7.4%, and 12.8%, respectively. GPB were isolated from all the surfaces sampled. A total of 94 GPB isolates were recovered, comprising Bacillus species (51.1%), Staphylococcus species (38.3%), Corynebacterium species (6.4%), and Enterococcus species (4.3%). Only 13.8% of the GPB were resistant to at least one of the biocides tested. Multiple drug resistance to at least three classes of antimicrobial agents was exhibited by 95% of the isolates tested. Conclusion: The results of this study suggest that veterinary clinic environments and surfaces in Makurdi are reservoirs of wide varieties of antimicrobial-resistant GPB of veterinary and public health importance.


Introduction
Poor disinfection, decontamination, and sterilization of hospital equipment and surfaces have been reported to be responsible for nosocomial infections in human healthcare settings and veterinary medicine.Contaminated hospital surfaces and equipment can be sources of infection because bacteria from the skin or feces of a patient could contaminate hospital surfaces and equipment and be transmitted from one patient to another if not cleaned and disinfected appropriately between patient uses [1][2][3][4][5].Among the consequences of hospital-acquired infections in human and veterinary medicine are increased morbidity and mortality, duration of hospitalization, and cost of treatment, often because the infective organisms are multidrug-resistant (MDR) [4][5][6][7][8][9][10].
Outbreaks of nosocomial infections have been reported in many veterinary teaching hospitals [4].Little is known about the prevalence of nosocomial infections in privately owned veterinary clinics [3,4].The vast majority of these nosocomial infections were estimated to be caused by bacteria, and the most often encountered species include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella species [1,11].Environmental contamination by Staphylococcus species [1,3,12] and Akwuobu et al. / Vet.Res.Notes., 3(11): 86-94, November 2023 Enterococcus species with multidrug resistance [4] is a source of infections in hospitals.
Some frequently used biocides have been reported to be contaminated with nosocomial Gram-positive (GPB) and Gram-negative bacteria (GNB) [17], which implies the development of resistance by these pathogens.The development of antimicrobial resistance in bacteria could also be attributed to the extensive use and misuse of disinfectants [13][14][15][16].
The paucity of documented reports on the roles of veterinary clinic surfaces as reservoirs for bacteria and sources of nosocomial infections in animals created a knowledge gap about the pathogenicity and antimicrobial susceptibility profile of these bacteria in Nigeria.Documented reports on the antibiotic vis-à-vis biocide susceptibility profiles of these pathogens are lacking for the veterinary hospital environment in Benue State.Therefore, the purpose of this study was to investigate the occurrence and antimicrobial susceptibility profile of Gram-positive aerobic bacteria from veterinary clinic environments in Benue State, Nigeria.

Sample collection and processing
Veterinary clinics I, II, III, and IV were the four clinics purposefully selected for the study in Makurdi, Benue State, Nigeria.The surfaces of examination/treatment tables (TTs), clinic floor, floor of the animal waiting area, clinician/receptionist desk, door handles, drug cabinet/table, chair hands, etc., were swabbed with sterile wooden handle swabs moistened in sterile peptone water.Each swab was put in peptone water that was appropriately identified and transported to the laboratory for processing.The broths were incubated at 37°C for 18-24 h.A loopful of the overnight broth cultures was subsequently streaked onto mannitol salt agar (MSA) and blood agar (BA) plates and incubated aerobically at 37°C for 24-48 hours.Resultant colonies, after incubation, were visually examined, and a single colony of each morphology on MSA/BA was sub-cultured to purity onto nutrient agar, and pure cultures were stocked out on nutrient agar slants and stored at 4°C until required for further processing.Standard microbiological procedures [18] were used to identify the isolates at the generic or species level.
As shown in Figure 1, high isolation rates were obtained from the surfaces of TTs, receptionist/clinician desks, drip stands and door handles, the floor of the animal waiting area, the drug cabinet and client chairs, the floor of the treatment room, and the weighing balance (WB).
Eighty-one (86.2%) of the GPB isolates were susceptible to all biocides, and 13 (13.8%)were resistant to at least one biocide tested (Table 2).The two isolates resistant to both purity and septal were Bacillus species (Table 3).
Table 4 shows the results of the biocide MICs of the 94 isolates of GPB.All the GPB tested were inhibited by dettol at varying concentrations, with the majority of the GPB having their MIC at 1.56 µl/ml.Septol inhibited 69 (73.4%) of the GPB, and most of the inhibited isolates have their MIC at 6.25 µl/ml.For purit disinfectant, 89 (94.7%) of the GPB were inhibited at varying concentrations.All the GPB were inhibited by tetmosol at varying concentrations, with most of the isolates having their MIC at 1.56 µl/ml.An isolate exhibited an ambiguous MIC for tetmosol.Also, all the   isolates were inhibited by Z germicide, and most have their MIC at 12.5 µl/ml.The results of the biocide MBCs of the GPB are shown in Table 5. Dettol and purit were bactericidal at concentrations ranging from 1.56 to 50 µl/ml, while tetmosol was bactericidal at concentrations ranging from 1.56 to 25 µl/ml.Septol and Z germicide demonstrated bactericidal actions on the isolates at concentrations ranging from 12.5-50 µl/ml and 12.5-25 µl/ml, respectively.
Table 6 shows the antimicrobial resistance profiles of GPB isolated in this study.High rates of resistance (31%-100%) were displayed by the isolates to 10 of the 12 antibiotics tested.With the exception of imipenem, isolates of Bacillus showed high resistance rates, ranging from 30% to 100%, to all the other antibiotics tested.Similarly, Staphylococcus species isolates were also resistant to penicillin, amoxicillin, cefoxitin, vancomycin, streptomycin, tetracycline, chloramphenicol, enrofloxacin, and sulphamethoxazole/trimethoprim.The isolates displayed low rates (5%-21%) of resistance to imipenem, gentamicin, and linezolid.High rates of resistance ranging from 25% to 100% were recorded for Enterococcus species to 11 antibiotics.The isolates of Corynebacterium species tested were resistant to only three antibiotics, viz., penicillin, amoxicillin, and streptomycin.
None of the GPB was susceptible to all the antimicrobials tested (Table 7).Table 8 shows the different resistance patterns recorded for the 55 GPB isolates tested.The researchers observed forty resistance patterns, with P + S + AML emerging as the predominant pattern.A wide spectrum of MDR was observed among 52 (95%) isolates used for the antimicrobial sensitivity test.

Discussion
The isolation of GPB, such as Staphylococcus, Enterococcus, and Corynebacterium species, in this study, shows that veterinary clinic environments in Makurdi, Benue State, are reservoirs of varieties of bacterial pathogens of veterinary and public health importance.Anyanwu et al. [22], in a similar study, reported Bacillus species (39.3%) and Staphylococcus species (32.5%) as the predominant surface contaminants of veterinary clinics in Enugu State, Nigeria.A review of health care-associated infections [1] revealed that around 80% of health care-associated infections were caused by 12-17 microorganisms.These microorganisms included, among others, Staphylococcus and Enterococcus  species.The retrospective study reported by Shoen et al. [23] on the analysis of Staphylococcus infections in the Oregon State University Veterinary Hospital pointed to the importance of S. aureus and S. pseudintermedius as hospital-acquired infections in veterinary hospitals.
The high isolation rate for TTs may be, as pointed out by Akwuobu et al. [19] in an earlier similar study, due to the examination and treatment of many different animals on TT, resulting in contamination of these tables with different types of microorganisms.If these tables are not properly cleaned and disinfected after each treatment and at the end of the work, they serve as a source of infection for other animals.
The high isolation rate of GPB from the surfaces of the receptionist/clinician desks could be due to the fact that these surfaces, which are frequently touched by the clinicians as they perform their duties, are rarely disinfected.WBs, by the nature of their function, are frequently used in clinics.In a previous study [19], it was opined that the high isolation frequency from WB may also be due to inadequate cleaning and disinfection of the contaminated surfaces of weighing scales after each use.The high rate of isolation   of bacteria from door handles in this present study also agrees with the reports of similar studies conducted in veterinary clinics in Makurdi [19] and in a tertiary care hospital in Nepal [24].This should be expected, as shown in these previous studies [19,24] because door handles are the most frequently touched surfaces in clinics that are neglected for cleaning and disinfection.The present study also revealed that client chairs are among the surfaces contaminated with potential pathogens of GPB.Despite being among the frequently touched surfaces in the clinics, client chairs are rarely, if at all, disinfected, as pointed out in an earlier report [19].
The high number of aerobic GPB isolated from the floors of treatment rooms and animal waiting areas in this study concurs with the report of Anyanwu et al. [22] in a study carried out in veterinary clinics in Enugu.Inadequate cleaning and disinfection of these areas may be attributed to the high number of aerobic GPB isolated from the floors of treatment rooms and animal waiting areas in the present study.In all the veterinary clinics visited for this study, these surfaces were cleaned only when an animal messed up the floors and at the end of work.This may suggest that the dilutions of the disinfectants used for the cleaning of these floors were ineffective against the contaminating GPB.Other frequently touched surfaces that are neglected for cleaning and disinfection in veterinary clinics include drug cabinets, drip stands, clinic switches, and clinic fridge door handles.This negligence could be the reason for the high isolation frequencies of aerobic GPB from these surfaces.
In the previous study in Makurdi [19], it was also revealed that handwashing basins recorded a high number of aerobic GPB isolates.This finding was surprising since this is the place where clinic staff wash their hands after attending to patients.This finding may suggest that either the soap and the disinfectant or the dilutions of the disinfectants used in these clinics for handwashing are not effective against these GPBs.Anyanwu et al. [22] reported similar findings in veterinary clinics in Enugu, Nigeria.The low rates of isolation of GPB from the surfaces of waste bin handles (WBH) observed in this present study parallel the findings in the previous similar study [19].In the previous study [19], it was opined that despite the fact that WBHs are one of the most frequently used items in clinics that are not considered for cleaning and disinfection, most users of these bins refrain from touching the handle.Some sampled items, such as the room divider (RD), animal bath (AB), and muzzle (M), were found only in veterinary clinic I. Thus, the number of such items sampled was far less than the number of other items found in all the clinics visited.This could explain the low isolation rates of GPB from the surfaces of these items.The previous study [19] also made a similar observation.
This study revealed that when Dettol, cytosol, and z-germicide are used at the dilutions recommended by the manufacturers, these biocides are very effective against aerobic GPB.In a previous study [19], similar results of high susceptibility to the cytosol (100%), z-germicide (100%), and Dettol (99.1%) were recorded against GNB.The efficaciousness of these disinfectants was attributed to their active components, such as chloroxylenol and isopropyl alcohol found in dettol and cytosol, which are collectively bactericidal with broad-spectrum antimicrobial activities.In addition to these components, tetmosol contains sodium hydroxide.Tar acid phenol and cresylic creosote are phenolic compounds that are active agents in z-germicide.These agents are also bactericidal with broad-spectrum antimicrobial activities.
The high rates of resistance by the GPB to septal and purity, in spite of using the recommended maximum concentrations of the disinfectants for domestic cleaning in this present study, were also reported in the previous study [19] with the GNB.Both the present study and the previous one [19], respectively, revealed that some GPB and GNB were resistant to these disinfectants at concentrations far above the recommended concentrations.The routine use of these disinfectants in clinics and households makes this issue particularly worrisome.The substitution of chloroxylenol with chlorhexidine gluconate in purity may account for the resistance of bacteria to the disinfectant, while the high resistance rate recorded for septal could be attributed to its composition (pine oils and 5-chloro-2-hydroxyl methane) and absence of any of the active agents found in the other more effective disinfectants.This study also corroborates the earlier report [19] that highlighted the ineffectiveness of pine oils and 5-chloro-2-hydroxyl methane as the only active agents in a disinfectant.The results of the present study may suggest that these biocide-resistant strains of aerobic GPB may be responsible for the contamination of some in-use disinfectants, as mentioned in the report of Al-Talib et al. [17].
The high rates of resistance to penicillin and amoxicillin displayed by GPB reveal the development of resistance to these β-lactams by strains of GPB in the study area.This is possible because GPBcan evolve different mechanisms for the inactivation of β-lactams.For example, resistance to these compounds in Staphylococcus and Enterococcus species is mostly achieved by modifications of their target sites, the penicillin-binding proteins, the production of β-lactamases, and selection pressures caused by inappropriate antibiotic stewardship practices [10,25,26].This enzyme confers resistance to penicillin, amoxicillin, and ampicillin.Though there are few studies of antibiotic resistance in Bacillus species, expression of β-lactamase genes has been reported in environmental isolates of B. subtilis [27].This could be the reason for the high rate of resistance recorded for Bacillus species in this study.
Similar to the results of other studies [22,28], the GPB isolates generally displayed a high level of susceptibility to imipenem.The high level of resistance to cefoxitin, a second-generation cephalosporin, may be due to mutations that result in alterations or loss of specific porin channels, thus reducing antibiotic concentrations [25].The isolates of staphylococci tested in the present study exhibited a high rate (58%) of resistance to vancomycin, a glycopeptide antibiotic that inhibits cell wall synthesis.This observation is a cause for alarm, as vancomycin has become the preferred agent due to the increasing resistance to β-lactams such as methicillin [29].This finding also indicates that these resistant strains can become endemic in the veterinary clinic environments where they can be transmitted.The antibiotic resistance of the enterococci isolated in this study may be attributed to their intrinsic resistance to many classes of antibiotics, driven by selection pressures resulting from misuse of antibiotics [10].Our finding agrees with the report of KuKanich et al. [4], where no enterococcal isolates from small animal veterinary hospitals were resistant to vancomycin.It is surprising that the GPB in this study exhibited a high level of resistance to gentamicin, which is rarely used for treatment in animals.Oral drug preparations containing aminoglycosides such as streptomycin and tetracycline long-acting in injectable forms are extensively used in veterinary medicine in Nigeria, and this may account for the high resistance to these antibiotics observed in this study.
The linezolid resistance level observed in this study was very low.Isolates of staphylococci demonstrated the lowest resistance rate (1/22, 5%) to linezolid, a class of synthetic bacteriostatic antibiotics called oxazolidinones with broad Gram-positive activity [26].Linezolid has potent in vitro activity against clinically important Staphylococcus species.
The resistance level to enrofloxacin in GPB is relatively high and calls for attention.In their study in Enugu, Anyanwu et al. [22] reported a low level of resistance to ciprofloxacin (a fluoroquinolone) in GPB.The indiscriminate use of sulfonamides in Nigeria could be responsible for the high resistance to sulphamethoxazole/trimethoprim, notwithstanding that it is a broad-spectrum sulfonamide.
MDR to at least three classes of antimicrobial agents exhibited by 95% of the isolates tested in this study was very high compared to similar studies in hospital environments and thus should be of public health concern.KuKanich et al. [4] reported 53% isolates of Enterococcus faecium from surfaces of equipment in small animal veterinary hospitals that were MDR to antimicrobial agents commonly used in veterinary medicine.In a similar study [24] conducted in a tertiary care hospital in Nepal, S. aureus isolates (29.5%) were also found to be MDR.
Our findings indicate that multiple drug-resistant bacteria have the tendency to develop resistance to biocides.This tendency was also demonstrated by GNB in the previous study [19].On the other hand, the development of multiple drug resistance in 88% of the biocide-resistant isolates in this study further supports the report of Kampf [30] on the development of antibiotic resistance in GNB after exposure to sublethal concentrations of some biocidal agents.

Conclusion
The isolation of multiple drug-resistant Staphylococcus, Enterococcus, Corynebacterium, and even Bacillus species in the present study calls for serious veterinary and public health attention, as veterinary clinic surfaces could serve as reservoirs of potential pathogens of GPB.Staphylococcus species are known to be a common cause of opportunistic infections in both humans and animals.Regular handwashing and cleaning of surfaces and environments using any of the effective biocides, viz., Dettol, cytosol, and z-germicide, are recommended.Other disinfectants containing the active agents found in these three disinfectants are also recommended for sanitation.Users should adhere to the instructions on the use of biocides.Compliance with instructions on the use of biocides, as given by the manufacturers, will contribute enormously to the mitigation of the development of antibacterial resistance in bacteria and the spread of MDR bacterial infections in humans and animals.

Table 1 .
Isolates of potential pathogenic bacteria from veterinary clinic environment.
a % of row total.b % of column total.

Table 2 .
Frequency of biocide susceptibility/resistance of bacterial isolates.
a % of row total.b % of total bacterial isolates (94).

Table 3 .
Frequency of occurrence of biocide resistance among GPB isolated from surfaces of veterinary clinics in Makurdi, Nigeria isolates.
a % of total isolates for each bacterial group.

Table 4 .
Frequency of biocide MIC for GPB isolated from surfaces of veterinary clinics in Makurdi, Nigeria.
a One isolate with ambiguous MIC; % = Percentage of total isolates tested.

Table 5 .
Frequency of biocide MBC for GPB.
a Percentage of total isolates tested.
a % of row total.

Table 7 .
Number of antimicrobial agents to which the bacterial isolates are resistant.

Table 8 .
Antibiotic resistance patterns of the GPB.