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¹Ogbomo RO, ¹Otene CI, ²Borke ME, ¹Oriarewo PO. ³Eifediyi RA, ⁴Inyang NJ, ⁵Agwu E

Addresses: ¹Surgery and ²Pathology Departments, Delta State University Teaching Hospital, Oghara, Delta State, Nigeria.

³Department of Obstetrics and Gynecology, College of Medicine, Ambrose Alli University, Ekpoma, Edo State, Nigeria.

⁴Department of Medical Laboratory Sciences Ambrose Alli University, Ekpoma, Edo State, Nigeria., ⁵ Microbiology Department, Faculty of Basic Medical Sciences, Kampala International University, Western Campus, Uganda.

Citation: Ogbomo RO, Otene CI, Borke ME, Oriarewo PO, Eifediyi RA, Inyang NJ, Agwu E. Emergence of Candida Species among bacterial pathogens recovered from surgical wounds in Delta State University Teaching Hospital, Oghara, Nigeria. Special Fungal Pathogens Journal, 2016, Vol 1, No 1 Pg 0021-0027.  Correspondence: [email protected]

Abstract

Background: Management of non-healing or delayed healing wounds remains a public challenge especially among patients with an underlying medical condition and with Candida prevalent polymicrobial etiology.

Objectives: To outline the polymicrobial nature of surgical wounds in resource-poor settings with an emphasis on Candida, and their implications in the design and implementation of effective intervention.

Material and methods:  A retrospective study of results of wound swabs sent for microscopy, culture, and sensitivity from June 2010 to May 2014 were retrieved from record books and analyzed. Information retrieved included demographic characteristics of participants, microbial population isolated, and antibacterial susceptibility of patients,  Standard method of isolation for recorded Candida and other organisms were obtained from the laboratory standard operating procedures and other laboratory diagnostic guidelines made available during the period covered by the study. Statistical significance of data generated was tested with SPSS version 20.

Results: The emergence of Candida species among the bacterial pathogens of wound infections shows changing patterns of disease epidemiology and it has implications for the effective intervention of chronic non-healing or delayed healing wound infection. The commonest bacterial organisms were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella species, and Proteus species in descending order.

Gram-negative enteric bacteria were the commonest open wound pathogens. Staphylococcus aureus is the commonest gram-positive organism found in open wounds. The least bacterial pathogen was Acinetobacter species and followed by Citrobacter species and Alkaligenes species. The commonest antibiotics that the bacteria were sensitive to were Ceftazidime, Gentamicin, Ofloxacin, Augmentin, and Cefotaxime.

Conclusion: The emergence of Candida species among bacterial agents shows a paradigm shift in routine and complicated wound management and has implications for effective intervention. Both gram-positive and gram-negative bacteria play active roles in the infection of wounds in our environment, the commonest organisms being Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

Key Words: Aerobic bacteria, Open Surgical wounds, Isolates.

Introduction

Fungi (Candida) of medical importance continue to participate in human diseases although many fungi (Candida) are opportunistic agents of infections, and preventive measures including chemotherapy are available for effective intervention.  Thus, they are among the four (bacteria, viral, and parasites) important agents of human disease including life-threatening invasive infections. They are very commonly diagnosed in HIV/AIDS and deep-seated systemic infections are often associated with patients with neutropenia.

The most common cause of Candidosis is the polymorphic species Candida albicans, which can grow as yeast cells, Pseudohyphae, and hyphae. It also produces chlamydospores which are refractile spore-like structures that are mainly produced at the termini of hyphae under specific environmental conditions in vitro. However, other Candida species, including Candida glabrata and Candida parapsilosis, are currently also significant human pathogens. Although the yeast Candida albicans remain a universal pathogen in humans, relatively little is known about the relationship between commensal and infecting strains, the origins of infecting strains, the transmission of the organism between individuals, and strain specialization

Surgical site infections (SSIs) are infections of the tissues, organs or spaces, exposed by surgeons during the performance of an invasive procedure 3. It can be caused by the degree of microbial contamination of the wound during surgery, the duration of the procedure, and host factors, such as diabetes, malnutrition, obesity, immunosuppression, and the number of other underlying disease states. (1). SSIs are associated with great illness and occasional lethality, as well as substantial healthcare costs and patient inconvenience and dissatisfaction (2)

Bacteria are responsible for the majority of surgical infections. Gram-positive bacteria that frequently cause infections in surgical patients include aerobic skin commensals and enteric organisms. Many pathogenic gram-negative bacterial species are capable of causing surgical infections, predominantly members of the Enterobacteriaceae family. Anaerobic bacteria, fungi, and viruses are the other causes of SSIs.  Bacteria species; including pathogenic and non-pathogenic bacteria, are ubiquitous (1). Aerobic bacteria are most commonly found in skin and open wounds as they contaminate, colonize, and cause skin infection. Less commonly present are viruses and fungi. Extrinsic and intrinsic factors which control microbial ubiquity abound and most are well understood.

A lot of effort in handwashing (3) procedures (sterile draping, scrubbing, use of facemask), techniques (irrigation, jet lavage, and meticulous dissection), and antibiotics have been developed to prevent Microbes from colonizing and infecting wounds but they still do when conditions that favor their growth and multiplication exist. Bacteria are found in intact skin, nasopharynx, mouth, air, surfaces, and soil.

When bacteria infect open wounds, they present more difficult challenges which may include delayed wound healing, chronicity of the wound, and scarring (4). Constant Microbial surveillance of wound infections is therefore imperative to generate a database for effective intervention. This study was therefore designed to retrospectively determine the fungal and bacteria load of surgical sites in Delta State University Teaching Hospital, Nigeria 

Material and Method

A retrospective study of results of wound swab sent for microscopy, culture, and sensitivity from June 2010 to May 2014 at the Delta State University Teaching Hospital which serves the tertiary health care needs of the people of the state and neighboring states of Edo and Bayelsa. It has many surgical specialties such as Orthopaedics, Burns/Plastic, Urology, Otorhinolaryngology Neurosurgery, Paediatrics Surgery, and General Surgery. The laboratory is one of the best in the country with international class Quality Assurance and Quality control.

 Sample collection and inclusion criteria

Five hundred and twelve (512) surgical site wound swabs were aseptically collected and analyzed at the Microbiology laboratory of Delta State University Teaching hospital, Nigeria.

 Analysis of samples

An aseptic and standard microbiological method contained in our former report (5) was adopted in media preparation and sample inoculation. MacConkey agar, Blood agar base, Nutrient agar, and Mueller Hinton sensitivity testing agar medium were prepared according to the manufacturer’s (OXOID Limited UK) instructions. They were sterilized at 121⁰C for 15 minutes holding time in an autoclave. Ten percent sheep blood agar was prepared by mixing 10 ml fresh sheep blood with 90 ml molten blood agar base at about 45⁰C. About 20 ml of each medium was dispensed on a sterile disposable plastic Petri dish and allowed to set. Samples were inoculated onto MacConkey, Nutrient, and Blood agar plates respectively for bacterial isolation. They were all incubated at 37±3⁰C for 18-24 hours.

Direct gram smear was made on a microscope slide and wet mounts prepared and examined microscopically. Significant mixed growth of bacteria colonies (more than 25 colonies per plate) was separated into single colonies by obtaining purity plates and all suspect colonies for Pseudomonas aeruginosa and Staphylococcus aureus were identified according to the scheme of Cowan and Steel’s Manual for the identification of medically important bacteria as revised by Barrow and Feltham (6). Duplicate samples were inoculated on Sabouraud Dextrose Agar for routine fungal isolation and yeast isolates were further subcultured in Chromogenic agar for further identification

Antibiotic susceptibility test

A commercially prepared antibiotic disc (Difco) was used. Kirby-Bauer’s Clinical Laboratory Standards Institute (7) modified disc diffusion technique for the antibiotic susceptibility test was adopted in this investigation. Control of test performance outlined by CLSI (8) was strictly followed. After incubation at 35⁰C for 18-24 hours, zone sizes were measured and interpreted using CLSI (8) standards. The criterion for antibiotic inclusion was based on first-line broad and narrow-spectrum antibiotics commonly used for soft tissue infection in the study area and in line with the guideline for antibiotic selection during susceptibility testing, by Clinical Laboratory Standards Institute (7-8).

Tabulation of the age, sex, hospital number, diagnosis, organisms found on microscopy, and culture from the records of the Pathology department of the hospital. Some of the wounds were excluded from this study and the exclusion criteria included; malignant wounds, neonates, pressure ulcers. The data was analyzed using SPSS version 20.

 

Results

The commonest organisms were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella, and Proteus in descending order. Gram-negative enteric bacteria were the commonest open wound pathogens. Staphylococcus aureus is the commonest gram-positive organism found in open wounds. The least bacterial pathogen was Acinetobacter and followed by Citrobacter and Alkaligenes. 23% of cultures and microscopy yielded no growth after 48 hours of incubation. 77% isolation rate. Candida albicans, a fungus was also isolated from open wounds in 0.6% of cases showing that fungi can contaminate, colonize and infect open wounds.

Table 1 shows the spectrum of organisms grown from open surgical wounds and the antibiotics sensitivity profile of isolated strains.  The organisms that had the highest number of growth were E. coli (14.6%) while the least was candida (0.6%). The antibiotics with the highest occurring sensitivity were CAZ (50.8%), followed by gentamycin (44.9%); while the antibiotics with the least sensitivity were CRF (1.3%).

Table 2 describes the association between organisms and age group. The antibiotic with the highest sensitivity was Citrobacter and this was seen among the 31 – 60 years age bracket; whereas no sensitivity was seen for Citrobacter among 61 – 90 years bracket. This difference is not statistically significant. (p = 0.08). Table 3 describes the association between organisms and gender. The highest sensitivity among the males was seen with candida (66.7%), while among the females it was Enterobacter (60.9%). This difference is  not statistically significant (P = 0.747)

 

Table 1. Prevalence and antibiogram of Candida species and bacterial agents of infection among the studied population

Spectrum of organisms

Organisms	Freq (n)	(%)			Sensitivity No (%)			Resistance No (%)
Acinetobacter	5	1.0			Aug	138	35.0	65%
Alkaligenes	24	4.7			CAZ	200	50.8	49.2
Candida	3	0.6			CRX	86	21.8	78.2
Citrobacter	24	4.7			Oflo	156	39.6	60.4
E. coli	75	14.6			Gent	177	44.9	55.1
Enterobacter	23	4.5			Cip	31	7.9	92.1
Klebsiella	54	10.5			CRO	14	3.6	96.4
Proteus	51	10.0			CTF	126	32.0	68.0
Pseudomonas	69	13.5			Eryt	45	11.4	88.6
Staph. species	66	12.9			CXC	32	8.1	91.9
Nil	118	23.0			CEF	116	29.4	70.6

 

Table 2:             Age specific distribution of microbial isolates

                                                                                Age groups

Organisms	1 – 30	31 – 60	>60
Acinetobacter	1 (20.0)	2 (40.0)	2 (40.0)
Alkaligenes	9 (37.5)	12 (50.0)	3 (12.5)
Candida	1 (33.3)	2 (66.7)	0 (0.0)
Citrobact	7 (29.2)	17 (70.8)	0 (0.0)
E. coli	24 (32.0)	34 (45.3)	17 (22.7)
Enterobacter	5 (21.7)	17 (73.9)	1 (4.4)
Klebsiella	16 (29.6)	33 (61.1)	5. (9.3)
Proteus	9 (17.6)	30 (58.8)	12 (23.6)
Pseudomonas	24 (34.8)	36 (52.1)	9 (13.1)
Staph	24 (36.3)	30 (45.5)	12 (18.2)
TOTAL	120 (30.4)	213 (54.1)	61 (15.5)
	x2 = 29.43		P = 0.080

Table 3: Sex-specific distribution of microbial isolates.

                                                Sex

Antibiotics	Male n (%)	Female n (%)
Acinetobacter	3 (60.)	2 (40.0)
Alkaligenes	13 (51.2)	11 (45.8)
Candida	2 (66.7)	1 (33.3)
Citrobacter	12 (50.0)	12 (50.0)
E. coli	36 (48.0)	39 (52.0)
Enterobacter	9 (39.1)	14 (60.9)
Klebsiella	33 (61.1)	21 (38.9)
Proteus	28 (54.9)	23 (45.1)
Pseudomonas	34 (49.3)	35 (50.7)
Staph	36 (54.5)	30 (45.5)
TOTAL	206 (52.3)	188 (47.7)
	x2 = 6.78		P = 0.747

Discussion of Candida studies

The main aim of health care provision is to manage health problems successfully for the benefit of clients. Effective intervention implies eliminating or reducing the root course of the sickness to the barest minimum. Unfortunately, this basic principle is difficult to achieve in resource-poor settings. Delayed healing or nonhealing wounds with microbial etiology remain one of the reasons for health-seeking behaviors of many people dwellings in developing country settings.

Bacterial continue to dominate such wounds (open and closed) in hospitals located in resource-poor settings due to underlying medical conditions and poor hygiene (9). The incidences of treatment failure and delayed wound healing continue to pose serious public health challenges to healthcare providers in tropical African countries constituting one of the most common reasons for patients to see a doctor in this region (10). Opportunistic infections of these wounds are currently, increasingly related to the weakened defenses of the patient’s peripheral tissues and other underlying medical conditions.

In this study, it is interesting to note the presence of Candida species among the bacterial pathogens isolated from the wounds. Common causes of fungal serious, and in some cases, fatal infections in patients with underlying chronic abnormality like cancer include Candida species, followed by Aspergillus, Cryptococcus, Histoplasma, Phycomycetes, and Coccidioides (9-16). Of the patients reviewed in the present study, five had malignancies including bladder, stomach, and colon carcinomas. Candida albicans were the fungi predominantly isolated from abdominal incision specimens. The relation between cancer and fungi seems to reflect the results of our study, as about half of the patients are of this group and the distribution of the isolates was similar to literature.

Other reports (12-14) revealed that: nine (2%) of the agents of surgical infections were yeasts: Candida albicans was isolated in 5 (55.6%), Candida tropicalis in 3 (33.3%), and Candida. glabrata in 1 (11.1%) specimen(s). Based on the medical characteristics of the patients, Cutaneous candida infection could indicate the presence of at least one predisposing factor such as immunosuppression and another underlying chronic disease which may closely be associated with fungal growth on a wound swab taken from hospital patients.

Predisposing factors impairing the immune system may therefore be detected in most patients whose laboratory results show fungal growth predominantly cutaneous candidal infections. It is therefore important for clinicians to be aware of fungal infections that may develop in this group under risk, particularly after surgery and all Candida species isolated from high-risk patients should be identified to species level, as nonalbicans strains are often associated with resistance to antifungal agents (9, 11, 16)

Again, cutaneous candida infections can be a cause of delayed wound healing, especially in surgical wounds treated with antibacterial ointments and occlusive dressings. Fungal invasion is associated with higher mortality independent of age, burn size, or inhalation injury. The fungal invasion was detected on average 16 days after injury in some reports. Yeast is one of the causes of necrotizing fasciitis after herniorrhaphy is exceptionally rare and an exhaustive literature search uncovered one additional documented case that was ultimately fatal.

Indiscriminate use of antibiotics (self-medication) probably due to lack of prophylactic antibiotics policy in resource-poor settings,  expensive medical services by private clinics, lack of facilities for laboratory confirmation of etiology, and corresponding susceptibility testing may also outline the emergence of Yeast agents in surgical wounds. Careful use of appropriate broad-spectrum antimicrobial agents may lead to successful treatment of clinically infected chronic wounds characterized by polymicrobial flora. (10-15)

On the other hand, we also recorded the bacterial etiology of the wounds studied. The result of this study contrasted with other studies where Staphylococcus aureus was the most commonly isolated pathogenic bacteria.  Staphylococcus being the most common organism on the skin, other surfaces and it seems natural to be in open wounds (17-19).

Mohamudamen Mana et-al (17), predominantly isolated Staphylococcus aureus followed by E.coli in 150 specimens. Lopinto Dessalegn and colleagues (18) had a sample size of 194 patients in a five-month study with a 71.1% isolation rate and the bacteria isolated in decreasing order were S. aureus, E.coli, Klebsiella species, Proteus species, and P. aeruginosa. In another study involving 354 specimens, an isolation rate of 70.4% (19) was recorded. The organisms isolated in decreasing order too were S. aureus, Klebsiella species, E. coli, Proteus species, and Pseudomonas species

In our study, E. coli, Pseudomonas, and Staphylococcus all ranked pretty high in frequency both individually and collectively however, our sample size is larger (512 cases), for a longer duration (4 years review) and 77% Isolation rate. It is known that acute open wounds have S. aureus as the more common isolate while chronic wounds have more of the endogenous (enteric) bacteria isolates (20).

Our study period being longer could have meant that we incorporated more chronic wounds and the lack of regular hospital surveillance could also have meant more hospital-acquired infections thereby tilting the bacteria count in favor of the likes of E. coli and Pseudomonas species respectively. It may also be argued that this may be an emerging trend in a society where people abuse antibiotics and present late to the hospital. Anaerobic bacteria isolates are expensive, tedious, and beyond the means of most Hospital laboratories (20). The 23% where the culture yielded no growth may not have been bacteria-free as they may have harbored anaerobes.

Presence of 1% Acinetobacter species Table (1), depicts the current world trend in which Acinetobacter species are the emerging tropical agents of infection responsible for most cases of hospitalization in resources limited setting and Acinetobacter species is also the leading agents of bacteria resistance among hospital-acquired infection (21). To be specific, the genus Acinetobacter represents one of many bacterial strains that are found to be resistant to all existing antibiotic classes as well as a prodigious capacity to acquire new determinants of resistance (Table 2) (21).

The observed 13.5% prevalence of Pseudomonas species Table 1, appears to be usually lower than the previous 41.% reported from other regions of south Southern Nigeria (10-11) and is extremely lower than reports from other parts of Africa and the world such as African and India and Iran: 33%, 36% and 73.1% (21-23). The reasons for these difference in prevalence is still being debated but may include:  decrease in the level of general hygienic measures, mass production of low quality antiseptic and medicinal solutions for wound treatment.

sub-inhibitory antibiotic concentration in wounds due to the absence of stringent regulation guiding the use of antibiotics (25). Again, Underlying medical conditions and quality of diagnostic facilities may play a vital role in up-regulating the prevalence of pseudomonas species especially the strains resistant to most conventional antibiotics used in developing country settings. (Table 2)

The observed 12.9% prevalence of Staphylococcus species lower than the previous 30% report from a neighing South-Southern Nigeria city of Ekpoma (11). A general reduction in the level of personal hygienic, use of a sub-lethal dose of low-quality antibiotics and antiseptic and also medicinal solutions for wound treatment including indiscriminate use of antibiotic concentration without prescription in wounds due to absence of stringent regulation guiding the use of antibiotics may explain the observed level of prevalence and persistence of Staphylococcal species (11).

Again, Underlying medical conditions and quality of diagnostic facilities may play a vital role in up-regulating the prevalence of Staphylococcus species especially the strains resistant to most conventional antibiotics used in developing country settings. (Table). The presence of other enterobacteria on the wound may signify poor hygiene and poor asepsis techniques, especially during wound management.

Many of the organisms are quite sensitive to a number of the common and popular antibiotics and we believe that with regular and consistent anti-microbial surveillance, infection control can be enhanced when these organisms are identified early and appropriate antibiotic therapy instituted. To be more specific; the observation of resistance to different types of routine high profile antibiotics such as quinolones, cephalosporins, and macrolides, and aminoglycoside antibiotics raises the stakes for the antibiotic era in the management of tropical diseases (26).

The usual plan is to treat diseases with affordable but highly effective antibiotics to create room for viable options when the routine antibiotics fail to treat the disease for which it was intended (10). Cephalosporins and quinolones are usually reserved for use as a second or third-line drug. Antibiotics with beta-lactam rings such as the cephalosporins have a high tendency to produce beta-lactamase and extended beta-lactamase while others have seen in this study mainly show resistance, by inhibition of protein synthesis in the microbial cells.

The survey was in teaching hospitals where the clients are relatively rich being able to afford their daily bread and medications when sick. However, this observation of microbial resistance to high-class antibiotics recorded in this study sets dangerous precedence to prevention and control of infections by rural community dwellers living in hard to reach areas with poor resources because healthcare providers may soon run out of options for disease treatment with antibiotics (11, 25).  Thus, if isolated wound microorganisms are already resisting the most available and expensive drugs, then prevention with available and affordable drugs becomes a great challenge.

Conclusions

Candida species isolation from surgical wounds is unique and underscores the need for inclusion of fungal regimen in the routine management of surgical wounds and cases of treatment failure attention should be paid to possible polymicrobial resistance and the treatment plan for treatment failures. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are the three commonest aerobic bacterial isolates in our practice and they are sensitive to common and available antibiotics. This knowledge will help a great deal in infection control and prevention.

Conflicts of interest: None

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