Journal of Bacteriology & Parasitology

In observations of many PCI (Parasitology Center, Inc.) patients over the years, we noted that many experienced GI symptoms but no parasites were detected from fecal samples provided. These cases were explained as possibly relating to "other pathogenic organisms, ex., pathogenic bacteria, that can cause symptoms comparable to those produced by typical parasites [1]. However, no actual bacteriological testing was done for verification.

In a cross-sectional study of 5,792 fecal specimens from 2,896 patients in 48 states and the District of Columbia, 32% were found positive for protozoan and helminth parasites during the year 2000 [2]. The most common parasites, in order of prevalence, were Blastocystis hominis, Cryptosporidium parvum, and Entamoeba spp. The first two species were subsequently studied in more detail by Amin [3,4,5,6]. A sizable proportion of patients without infections, nevertheless, exhibited GI symptoms, including but not limited to diarrhea, constipation, and abdominal cramps, similar to those observed in parasite infected patients. Those patients remained unaccounted for in terms of causation.

Our present results verify the original assumption [1], document the identity of bacterial agents involved in the GI symptomology in patients proven to have no intestinal parasites, and provides the results of sensitivity and resistance tests for treatment purposes. The GI symptoms in those parasite-free patients can now be explained by the pathogenic bacteria documented for each case. A more recent study [7] shows that IBS associated with abdominal pain, bloating, and diarrhea is caused by intestinal bacteria.

Patients with GI symptoms who initially submit fecal specimens for Comprehensive Stool Analysis (CSA) only are encouraged to follow up with bacterial testing by inserting the following statement in their CSA diagnostic report: "Patients with symptoms but with no detected parasites are highly recommended to do the PCI swab culture test for pathogenic bacteria that cause GI symptoms similar to those caused by parasite infections. Swab kits are available by calling PCI at 480-767- 2522".

The study population constituted 60 patients (21 males and 39 females) between the ages of 2 and 87 yr who experienced GI symptoms, tested negatively for parasites using the PCI CSA, and tested for pathogenic bacteria at the same time, between August and December, 2010.

The CSA was performed [2] as follows. The specimens were collected and fixed in SAF, processed and stained in CONSED^™ according to manufacturer's (Alpha-Tec Systems, Inc., Vancouver, WA) directions. Fixed specimens were filtered, mixed with CONSED^™ and ethyl acetate, vortexed, and centrifuged. All but the fecal plug was decanted, and mixed with CONSED^™ diluting reagent. The plug was then transferred to and mounted on a microscope slide for light microscopy examination. All microscopic evaluations and identification were made by the same observer(s) blinded to patient information, e.g., symptoms, travel, etc. Positive results were quantified (number of organisms per high-power field on a scale of 1 to 4) from duplicate samples from the same patient.

Swabbed fecal specimens were collected using sterile transporter swabs manufactured in Italy by Copan for Healthlink, McKesson, Richmond, VA. Specimens were deposited in selective Platin Medium (MacConkey, XLD, SS agar) and incubated for 24-48 hr at 37^°C. Colony morphology was observed after Gram-staining. Suspected bacterial pathogens were then tube-tested using BIOQUIMICS test in Tse, Lia, Ornitin, Indol, and Simmon citrate, and incubated for 24 hr at 37^°C or tested using packed BIOQUIMICS. Specimens were then identified using criteria of colony morphology of common enteric bacteria on differential and selective plating media, e.g., MacConkey agar with crystal violet, XLD agar, SS agar, DCA, and HEA [8].

Sensitivity results were obtained by culturing identified specimens in Mueller-Hinton agar. Antibiotic discs for Gram-positive and Gramnegative bacteria were then placed on the culture and sensitivity and resistance results were read. The disc antibiotic concentrations varied between 30.0 µg, 10.0 µg, 5.0 µg, and 1.0 µg for various antibiotics tested.

Five species of gram negative pathogenic bacteria and 1 fungal species were cultured from fecal swabs of a random sample of 60 symptomatic, but parasite-free, patients tested between August and December, 2010. These are Escherechia coli (Migula, 1895) Castellani and Chalmers, 1919, Klebsiella sp., Proteus vulgaris Hauser, 1885, Citrobacter freundii (Braak 1928) Werkman and Gillen 1932, Pseudomonas aeruginosa (Schröter, 1872) Migula, 1900, and the fungus Candida sp. Most patients were concurrently infected with 2 or 3 species of bacteria (Table 1). The prevalence rate of the above cultures was 100 % in E. coli, 72% in Klebsiella sp., 33% in P. vulgaris, 25% in C. freundii, 7% in P. aerruginosa, and 5% in Candida sp. Of a total of 145 positive findings, E. coli made up 41% of total infections, Klebsiella sp. 30%, P. vulgaris 14%, C. freundii 10%, P. aeruginosa 3%, and Candida sp. 2%.

Table 1: Prevalence of infections with pathogenic bacteria in fecal specimens of 60 symptomatic patients free of between August and December, 2010 intestinal parasites sampled.

Diagnostic test results of patient cultures are supplemented with sensitivity and resistance test results derived independently for each individual patient's sample. Sensitivity test results are provided in 4 major categories with the most efficient antibiotics included in category no. 1 and the least efficient in category no. 4. A selection of antibiotics from categories 1 and 2 is recommended. Antibiotics in all categories are individually designed for the treatment of concurrent infections that the patient may have, and will not be the same from patient to patient even with the same infections. Antibiotics to which cultured species are resistant also varied from patient to patient.

The frequency distribution of Enterobacteriaceae associated with bactermia in the USA (courtesy of Barnes Hospital, St. Louis) was 45% for E. coli, 24% for Klebsiella, 9% for Proteus, and 4% for Citrobacter, among others (Murray et al., 1990). Our results were very similar for E. coli (41%) but slightly higher for Klebsiella sp. (30%) and P. vulgaris (14%), and C. freundii (10%). The Barnes Hospital survey included 2 other pathogens that were not found in our survey, Enterobacter sp. (13%), and Serratia sp. (4%) (Murray et al., 1990) compared to our finding of P. aerruginosa (3%) and Candida sp. (2%) made up the difference in the overall prevalence.

Most of our patients were infected with 2 or 3 species of bacteria. Their symptoms, thus, express the composite effect of their over all infections and cannot be assigned to single species of bacteria alone.

Escherechia coli

Over 700 antigenic serotypes of E. coli are recognized based on O, H, and K antigens. Most human beings have more than 1 strain of E. coli at the same time [9]. Most strains of E. coli live in the intestine of humans and other mammals without causing any pathology [10]. Pathogenic strains of E. coli, however, are responsible for 3 types of infections in humans: urinary tract infections, neonatal meningitis, and intestinal diseases. The latter includes (1) ETEC (Enterotoxigenic E. coli) causing diarrhea in infants and travelers, (2) EIEC (Enteroinvasive E. coli) causing dysentery-like diarrhea with fever, (3) EPEC (Enteropathogenic E. coli) causing watery, sometimes bloody, diarrhea especially in children, and (4) EHEC (Enterohemorrhagic E. coli) causing hemorrhagic diarrhea and/or food poisoning which may develop into hemolytic uremic syndrome (HUS) and includes the invasive 0157:H7 strain making up 80% of the EHEC serotypes producing the verotoxin or Shiga toxin [9,11]. We do not know which strain(s) of E. coli did our patients test positive for. Strain identification requires molecular techniques not readily available in most diagnostic laboratories. Our study population comprised patients of all age groups (2-87 yr old) and both sexes. Judging by symptoms alone, patient no.17 may have been experiencing an infection with an EPEC or EHEC strain.

Klebsiella sp.

Pathogenic varieties of Klebsiella are grouped in 2 antigenic groups: the O antigen with 9 varieties and the K antigen with over 80 varieties. Klebsiella is increasingly reported as a nosocomial infection second only to E. coli in urinary tract infections in women [12]. Klebsiella pneumoniae is an opportunistic infection in older patients with weakened immune system which also causes nosocomial pneumonia, intraabdominal infections and intestinal pathology. It is a resident of the intestinal track in about 40% of man and animals [13]. Klebsiella sp. was the second most commonly cultured bacteria in our study. It was not possible to assign any symptomology specific to Klebsiella alone since all Klebsiella infected patients in our study group were also concurrently infected with E. coli.

Proteus vulgaris

Proteus vulgaris inhabits the intestinal tract of humans and animals. It is also found in the soil, water, putrefied meat, and fecal matter and is associated with long-term care facilities and hospitals where it is also known to colonize the skin and oral mucosa of patients and hospital personnel alike. It is an opportunistic pathogen in humans where it is also known to cause urinary tract (UT) and wound infections [14,15]. While Proteus spp. are not the most common sources of bacterial infections in humans, P. vulgaris holds yet a smaller role in the pathology caused by this group [16]. Proteus species most frequently cause UT infections, with Proteus mirabilis Hauser, 1885 producing 90% of the cases [14,15]. In our study population of 60 patients, P. vulgaris infected mostly 19-58 yr old females; only 7 males were infected. It is, herein, suggested that the prevalence of P. vulgaris infections in the intestinal tract of females (Table 1) may be related to cross contamination from UT infections not tested for in the same patients. In our study population, it infected 7 males and 13 females.

Citrobacter fruendi

Citrobacter is found in the human intestine and almost everywhere else including water, waste water, soil, etc. It is an indicator of a potential source of contamination but is rarely a source of illness [17]. Citrobacter freundi, however, is often the cause of opportunistic infections mostly causing abnormal inflammatory changes in the intestinal tract [17] and affecting biliary, urinary, and respiratory tracts, and blood of patients with weak immune system [18]. It has been "suspected to cause diarrhea and possibly extraintestinal infections including peritonitis [19]. Of 38 hospitalized patients in 2 community teaching hospitals in the Detroit Medical Center, Citrobacter bacteremia developed in elderly patients (65%) and was hospital acquired (77%) with initial sites of infection including the UT (39%), intestinal tract (27%), wound (10%), and unknown (13%) [17]. In our patient population, it infected 5 males and 10 females.

Pseudomonas aeruginosa

Pseudomonas aeruginosa is a free-living organism commonly found in soil and water as well as on the surfaces of plants and animals. It is an emerging opportunistic and nosocomial pathogen infecting only compromised tissues and causing pathology in thegastrointestinal tract, heart, blood, respiratory system, central nervous system, ear, eye, bone and joint, UT, skin, and soft tissues. In the intestinal tract, it causes pathology from the oropharynx to the rectum including perirectal infections, pediatric diarrhea, typical gastroenteritis, and necrotizing enterocolitis. The GI tract is also an important portal on entry in Pseudomonas septicemia and bacteremia. It has been isolated from the throat (5%) and stool (3%) of nonhospitalized patients. In some studies, gastrointestinal carriage rates increased in hospitalized patients to 20% within 72 hr of admission [9].

Four patients, (7%) of our study population of 60 were infected with P. aeruginosa. All were concurrently infected with E. coli and P. vulgaris and experienced mixed GI symptoms. Four other patients of 25 who were not tested for parasites (16%) were also infected with P. aeruginosa but their symptoms were not known in the absence of CSA test results.

Candida sp.

Fecal specimens of only 3 patients cultured positively for Candida concurrently with other infections (Table 1). Those patients were also positive for Candida using microscopical CSA. Diagnostic microscopical examination of fecal specimens of 40 other patients of the same study group of 60, using CSA, were positive for Candida at levels of 1 or 2 out of 4 possible infection intensities. This suggests that Candida, a fungus, does not readily grow in swab cultures and that microscopy provides a better detections in fecal specimens. The cyclical nature of Candida presence affected by diet and time of sampling after a compromising meal may be related.

Concurrent infections

As indicated earlier, most patients were concurrently infected with 2 or 3 species of bacteria (Table 1). It is clear that patients' symptoms are related to the cumulative effect of their composite infections that cannot be attributed to single bacterial species alone. IBS is one such situation [7] where the phenomenon of multiple causation applies.

We are grateful for Dr. Jesus Jimenez Salazar for technological help with the swab culture tests of fecal specimens. This investigation was supported by an Institutional Grant from Parasitology Center, Inc. in accordance with our new policy of encouraging patients to submit both CSA and swab culture tests to better understand factors involved in GI symptomology.