Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Evaluation of Resistant Urinary Tract Infections by Gram-positive Bacteria in Medina, Saudi Arabia

Sahar Saad Allah Amin, Nada A. Abdel-Aziz, Wael S. El-Sayed, Manal Ibrahim Hanafi Mahmoud, Eman Mohamed Sharaf Elsayed, Rehab A. Eltahlawi*

Department of Microbiology and Immunology, College of Medicine, Taibah University, Kingdom of Saudi Arabia

Abstract: Background: Gram-positive uropathogens have become common, associated with serious underlying illnesses and increasingly resistant to available antibiotics. Objectives: The goal of this study was to investigate the incidence and risk factors of gram-positive cocci UTIs in hospitalized patients in Medina, KSA and their susceptibility patterns to widely used antimicrobial agents. Methods: During a 12-month study, 165 clinical isolates of gram-positive cocci were recovered from 1137 culture-positive urine specimens at a tertiary hospital. Antimicrobial susceptibility of gram-positive cocci isolates was tested with the disk diffusion and E test methods. Molecular typing of some VRE isolates was done to detect the predominant Van genotypes. Results: Out of 8600 reviewed cases, 1137 (13.2%) were culture positive, 165 cases (14.5%) were gram positive cocci. E.faecalis formed 53.3% (88/165) of isolated gram-positive cocci, followed by E faecium (17.6%), S.agalactiae (23.6%) and S. aureus (5.5%). Multidrug resistant positive cocci formed 9.7% of gram-positive isolates including VRE (8.5%) and MRSA (1.2%). 75% of E. faecalis and 50% of E. faecium isolates were sensitive to nitrofurantoin, all VRE strains were sensitive to linezolid. All S. aureus isolates were sensitive to cefazolin, nirtofurantoin, sulfamethoxazole and vancomycin. 75% of MRSA strains were sensitive to sulfamethoxazole and all were sensitive to vancomycin. All isolates of S. agalactiae were sensitive to cefazolin and nitrofurantoin, and all were resistant to Trimethoprim-sulfamethoxazole. Van B genotype was detected. Conclusion: Vancomycin and nitrofurantoin seem to be effective drugs for treatment of gram positive UTIs. vanB genotype was detected.

Keywords: urinary tract infection, gram positive bacteria, antibiotic resistance, genotyping

Received February 03, 2021; Revised March 09, 2021; Accepted March 18, 2021

Citation: Sahar Saad Allah Amin, Nada A. Abdel-Aziz, Wael S. El-Sayed, Manal Ibrahim Hanafi Mahmoud, Eman Mohamed Sharaf Elsayed, Rehab A. Eltahlawi, Evaluation of Resistant Urinary Tract Infections by Gram-positive Bacteria in Medina, Saudi Arabia. American Journal of Microbiological Research, 9 (1). 14-24. January 2021. doi:10.12691/ajmr-9-1-3

1. Introduction

UTI refers to the presence of urinary tract microbial pathogens and is characterized as the growth of a single pathogen from properly collected mid-stream urine specimen of > 10⁵ colony-forming units per milliliter [1,2].

UTIs account for more than 100,000 hospital admissions annually, most often for pyelonephritis [3]. They also account for at least 40% of all hospital-acquired infections and are, in most cases, catheter associated [4]. Bacteriuria develops in up to 25% of patients who require a urinary catheter for > 7 days, with a daily risk of 5% [5]. Furthermore, bacteria, including, are continously exposed to the selective pressure from antibiotics or antiseptic agents in the nosocomial environment. Approximately 25% of the patients who have had an episode of acute cystitis clinically manifest recurrence [6]. Basically, most acute UTIs are confined to the lower urinary tract, but persistence and recurrence of the infection might lead to renal progression, producing pyelonephritis which can cause kidney damage or even failure if left untreated for an extended period of time [7].

UTIs during pregnancy affect mothers and fetus, causing premature labor, preeclampsia, anemia and transient renal insufficiency, intrauterine growth retardation (IUGR), and low birth weight. Nonetheless, not everyone with UTI (i.e., having a significant number of bacteria in urine) would necessarily develop recognizable signs and such a condition is defined as asymptomatic bacteruria (ASB) [8].

Relative frequency of the pathogens varies depending upon age, sex, catheterization and hospitalization. Escherichia coli and other Enterobacteriacae are the main causative agents of UTIs. Other gram-negative rods (Pseudomonas spp) and gram-positive cocci (coagulase-negative Staphylococci, Staphylococcus aureus, Streptococcus group B, Enterococci) are comparatively more common in some hospitalized patients, however [9].

Nosocomial UTIs comprise perhaps the largest institutional reservoir of nosocomial antibiotic resistant pathogens (Maki et al. 2001). Gram positive cocci isolates have demonstrated a remarkable capacity to rapidly establish antibiotic resistance over the past decade. Area-specific monitoring studies are required to identify trends of antimicrobial resistance, to implement successful treatment and to reduce mortality rates [10].

2. Subjects, Materials and Methods

2.1. Study Design, Period and Area

A prospective cross-sectional study was conducted on UTI cases attending Maternity and Child Hospital, Medina, Kingdom of Saudi Arabia, from April to June 2016.

2.2. Study Subjects

The research subjects who did not obtain antimicrobials during the previous seven days gave a total of 8600 clean catch midstream urine samples. Study subjects included catheterized patients more than 72 hours admitted to Medical, Surgical, Gynecology, and Maternity wards and non-catheterized patients from the outpatient departments with symptoms of UTI.

2.3. Sample Collection, Handling, and Transport

A freshly voided midstream urine samples (10-20 ml) were collected from non-catheterized patients in wide mouth sterile containers. Catheter urine specimens (10-20ml) were transferred to sterile containers after cleansing the outlets of the catheters with appropriate disinfectant. The urine specimens were delivered to the laboratory to be processed within one hour. If a delay was suspected, boric acid was used as a preservative (0.1 g/10 ml of urine).

2.4. Culture and Identification

Standard microbiological techniques are used in the culture of all urine specimens and in the identification of the isolates.

The uropathogens were isolated by a surface streak procedure on both blood and CLED agar (cysteine lactose electrolyte deficient medium) (Oxoid, Ltd., Basingstoke, Hampshire, England) by using calibrated loop (0.001/ml). Cultures were incubated in aerobic atmosphere at 37°C for 24 hrs. A positive urine culture was defined as colony count > 10⁵ CFU/ml for mid-stream urine and > 10² CFU/ml for catheter urine. Gram positive isolates were identified using catalase test, bile esculin test and phenotypic API strept, API staph methods according to the manufacturer’s instructions (BioMerieux, Marcy L'Etoile, France). Isolates were identified by the Vitek2 system [11].

2.5. Preparation of Stock Cultures

Stock cultures are prepared of all Gram-positive isolates using glycerol as the osmotic protector. 8 to 10 colonies from an overnight incubated plate are inoculated in 1 ml freeze broth (500 µl 30% glyecrol +500 µl broth). The broth was homogenized on a vortex mixer and stored at -70°C. Control plates are inoculated and incubated at 37°C overnight to check for contamination. If the control plate shows contamination, the strain is purified by reincubating it on CLED plate.

2.6. Antimicrobial Susceptibility Testing of Uropathogens

The antimicrobial susceptibility testing of Gram-positive isolates was done by the standard disk diffusion method of the Clinical and Laboratory Standards (13). The antibiotic discs and their concentrations were ampicillin (10μg), Cefazolin (30μg), ceftriaxone (30μg), norfloxacin (5μg), gentamicin (10μg), erythromycin (15μg), nitrofurantoin (300μg), TMP-SMX (25μg), and vancomycin (30μg). All the antimicrobials used for the study were obtained from Oxoid Ltd. Bashingstore Hampaire, UK. Standard inoculae adjusted to 0.5 McFarland were spread with swabs on the surface of Muller- Hinton agar plates (Oxoid Ltd. Bashingstore Hampaire, UK); antibiotic discs were dispensed after drying the plates which were then incubated for 24 hours at 37°C.

Reference strains of Enterococcus faecalis (ATCC 29212) and S. aureus (ATCC 29213), sensitive to the antimicrobial agents tested were used as a quality control throughout the study. Multi-drug resistance was defined as resistance to three or more of the antimicrobials tested [12].

2.7. E-test

E-test (bioMérieux) was used to determine the minimum inhibitory concentration (MIC) of linezolid. The test was performed on Mueller Hinton agar (Oxoid, Basingstoke, UK) with incubation for 16-18 h at 36°C. Susceptibility testing was performed, and breakpoints were established according to the recommendations of the CLSI. Resistance to oxacillin is measured by E- test using species-related breakpoints with resistance defined as an MIC of ≥ 2.0 μg/ml [13].

2.8. Vancomycin Resistance Gene Typing by PCR

Enterococci were first incubated overnight at 37°C in Todd-Hewitt broth, and then 1-ml volumes were microcentrifuged and the pellet was resuspended in 200 ml of TE buffer (10 mM Tris-HCl,1 mM EDTA [pH 8.0]). The suspensions were heated at 95°C for 20 min and then microcentrifuged for 2 min. Five-microliter volumes of the supernatant were subjected to PCR amplification in 50-ml reaction mixtures containing each deoxynucleoside triphosphate at a concentration of 200 mM, each primer at a concentration of approximately 1 mM, and 1 U of Taq polymerase (Boehringer Mannheim) in 10 mM Tris-HCl (pH 8.3)-50 mM KCl-2 mM MgCl2-0.1% gelatin-0.1% Tween 20-0.1% Nonidet P-40.

The samples were subjected to 35 PCR cycles, each consisting of 1 min of denaturation at 94°C, 2 min of annealing at 60°C, and 2 min of elongation at 72°C. PCRs were analyzed by electrophoresis on 2% agarose gels and were stained with ethidium bromide. The oligonucleotide primers used for detection of vanA, vanB, vanC1, and vanC2 or vanC3 sequences were designed with reference to the sequences deposited in GenBank by P. Courvalin and colleagues under accession numbers X56895, L06138, M75132, L29638, and L29639, respectively. Primer sequences and specificities are presented in Table 1.

For each sample, two PCRs were set up. One contained primers VanABF, VanAR, and VanBR, which direct amplification of 231- and 330-bp fragments from the vanA and vanB genes, respectively. The other contained primers VanC1F, VanC1R, VanC23F, and VanC23R, which direct amplification of 447- and 597-bp fragments from the vanC1 gene and either the vanC2 or the vanC3 gene, respectively. Multiplex PCRs were tested in duplicate. Known positive and negative controls were also included with each PCR run [14]. Genotype-negative VRE isolates, for which vancomycin MICs were 4 mg/ml, were also tested for the presence of vanD by using the primers described by Perichon et al. 1997 [15].

2.9. Statistical analysis

The statistical analyses will be performed using Statistical Package for the Social Sciences (SPSS for Windows Version 22.0). All the results will be tabulated and presented by figures and histograms using the one-way ANOVA repeated measures, and paired T-test.

Table 1. PCR primer sequences

3. Results

Out of 8600 reviewed cases, 1137 (13.2 %) were culture positive. Gram-positive cocci represented 165 (14.5 %) whereas 972 (85.5 %) were gram-negative bacilli. (Figure 1)

Figure 1. Distribution of the isolated organisms from urine

Escherichia coli (E-coli) formed 63.8% (620/972) of isolated gram-negative cultures followed by Enterobacter spp (18.5%) and Kelebsiella spp (13.4%), while Acinetobacter species were the least isolated among gram-negative organisms (only 8 isolates). (Table 2)

Table 2. Distribution of isolated organisms from urine

Out of 165 Gram positive cocci culture, the isolation rate of Enterococcus spp was 70.9% (117/165). Enterococcus faecalis was isolated from 88 cultures (53.3 %) whereas Entercoccus faecium was isolated from 29 cultures (17.6 %). Streptococcus agalactiae represented 39 isolates (23.6 %) then Staphylococcus Aureus 9 (5.5 %) isolates. (Table 2)

Frequency of isolated gram-positive cocci were higher among children (109/ 165) (66%) than adult females (56/165) (34%). The frequency of different gram-positive spp isolated from children and adult females are represented in Table 2. Enterococcus faecalis isolated from children were 62 out of 109 (56.9 %) and 26 out of 56 in adult females (46.4%) while Staphylococcus aureus was the least isolated organism in children (3/109) (2.7%) and Enterococcus faecium was the least isolated organism among adult females (1/56) (1.8%) (Figure 2).

Regarding children sex and age distribution, no significant difference in the frequency of isolation of gram-positive cocci from urine of male children (57/109) compared to female children (52/109) aged from 1 month to 13 years (52.3% and 47.7% respectively). However, gram-positive cocci were isolated from 28 infants ≤ 2 years and percentage of gram positive UTIs was significantly higher in male (71.4% “20/28”) than female (28.6% “8/28”) infants (Figure 3). Among children those with isolated gram-positive cocci from their urine, 41 (37.6%) were presented with only urinary tract infection while the rest of cases (68/109) (62.4%) presented with other clinical manifestations and anomalies associated with urinary tract infections including chest infections, polycystic kidney, hydronephrosis, sepsis, epilepsy, colitis and sickle sick anaemia (Figure 4). The majority of paediatric cases (55%) were admitted in inpatient paediatric departments, while 36.7% were from paediatric emergency department and 6.4% in neonatal intensive care units. Minority of cases were from other departments as nephrodialysis and behaviour and growth units (Figure 5).

Figure 2. Frequency of gram-positive isolates in children (A) and adult females (B)

Figure 3. Age and sex distribution of gram-positive urinary isolates among children

Figure 4. Clinical presentation of children with gram-positive urinary isolates (*chest infections, polycystic kidney, hydronephrosis, sepsis, epilepsy, colitis and sickle sick anaemia)

Figure 5. Distribution of children with gram-positive urinary isolates in hospital (* other departments as nephrodialysis and behaviour and growth units)

Figure 6. Clinical presentation of adult females with gram-positive urinary isolates (*premature rupture of membranes, vaginitis, vaginal bleeding and fever)

Figure 7. Frequency of multidrug resistant gram-positive isolates

Regarding adult females, 42 out of 56 (75%) diagnosed after urine culture as urinary tract infection were pregnant. 19 of those pregnant females presented as asymptomatic bacteruria (GBS were isolated from 9 out of 19 cases (47.4%) of asymptomatic bacteruria). The remaining 25% (14/56) of females included in the study presented with UTI associated with premature rupture of membranes, vaginitis, vaginal bleeding or fever (Figure 6).

Multidrug resistant positive cocci formed 9.7% (16/165) of total gram-positive isolates (Figure 7). VRE formed 87.5% (14/16) of multidrug resistant positive cocci, while MRSA formed 12.5% (2/16) (Figure 8).

Figure 8. Distribution of multidrug resistant gram-positive cocci

Regarding cases of multidrug resistant positive cocci, 62.5% (10/16) was males, 93.7% (15/16) aged less than two years, and 87.4% (14/16) admitted in wards as inpatients. Only one third (31.2%, 5/16) presented with only urinary tract infection meanwhile the other two thirds (68.8%, 11/16) presented as combination of urinary tract infection and other presentations as respiratory tract infection (45.5%, 5/11), sepsis and vomiting (18.2%, 2/11 each). Nearly all cases were admitted for less than 25 days (93.7%, 15/16) and was cured (93.7%, 15/16). (Table 3).

Table 3. Description of multidrug resistant gram-positive cocci cases

Figure 9. Antibiotic sensitivity

Figure 10. Agarose gel electrophoresis showing positive amplification of 330 bp fragment specific for Van B of VRE from clinical specimens, lane 1: size marker (100 bp DNA ladder), lan2 2: negative control, lane 3: positive control, lane 5: E. faecium VRE isolated from urine

Regarding antibiotic sensitivity results for isolated gram-positive cocci, all Enterococcus faecalis isolates were sensitive to ampicillin, vancomycin and linezolid while, 75% of them were sensitive to nitrofurantoin and 18% were sensitive to gentamycin. All Enterococcus faecium isolates were sensitive to linezolid, 50% of them were sensitive to nitrofurantoin drug and all were resistant to ampicillin. Data from our investigation showed varying susceptibility to antibiotics with significant resistance of Enterococcus spp isolates (more than 80%) to erythromycin, ampicillin, cefazolin, ceftriaxone, norfloxacin, and sulfamethoxazole-trimethoprim. VRE isolates were all resistant to vancomycin and all sensitive to linezolid drugs. All staph aureus isolates were sensitive to cefazolin, nirtofurantoin and sulfamethoxazole drugs while, all MRSA isolates were resistant to penicillin, ampicillin, cefazolin, and nitrofurantoin. 75% of MRSA isolates were sensitive to sulfamethoxazole and all were sensitive to vancomycin drugs. All isolates of streptococcus agalactiae were sensitive to cefazolin and nitrofurantoin drugs, while 52% of them resistant to ampicillin and all were resistant to sulfamethoxazole drugs (Figure 9). Molecular typing of VRE strain in this study revealed vanB genotype.

4. Discussion

Bacterial infection of the urinary tract is one of the common causes for seeking medical attention in the community. Effective management of patients suffering from bacterial UTIs commonly relays on the identification of the type of organisms that caused the disease and the selection of an effective antibiotic agent to the organism in question [16].

In this study, the isolation rate of bacteria from urine was 13.2 %. This finding is in line with a study done in Addis Ababa where the prevalence of urinary tract infection was 48/414 (11.6%) [17] and one from Iran which had a UTI rate of 13.2% [18]. This is possibly because UTI symptoms are not a reliable indicator of infection and in children younger than 2 years of age are non-specific.

In our investigation, most of the urine samples were collected from patients who did not have classic UTI symptoms, and most of the subjects had been referred by general practitioners not specialist physicians. These results indicate that urine culture is necessary in the majority of cases for a definitive diagnosis of UTI. On the other hand, our result is comparatively lower than reports within the country and other parts of the world. A study conducted in at the King Fahad National Guard Hospital in Riyadh reported 24.4% prevalence of hospital acquired UTI [19]. In another study in Kenya, 24% prevalence of UTI was detected [16], this might have been either due to sample size variation or the studies might have been based on retrospective survey.

In this study, the most frequent uropathogens were Gram negatives which made up to 85.5% of all isolates (Figure 1). Our result is in concordance with that published by Angoti et al, 2016 [20] who isolated gram negatives from 80% of positive urine cultures. E. coli was by far the most common bacteria isolated from urine samples in both outpatients and inpatients of both sexes (63.8%), followed by Enterobacter spp (18.5%), Klebsiella spp (13.4%) and Proteus spp. (5.2%) while Acinetobacter spp were the least isolated among gram-negative organisms (only 8 isolates i.e., 0.8%) (Table 2). This finding was in agreement with other studies [18,21] where Escherichia coli was the most common etiological agent of UTI (74.6%, 80.5% respectively), followed by Klebsiella spp (11.7%) (18) and Proteus spp. (6.1%) [21].

Recently, gram-positive species recovered from urine have gained special attention and are identified more frequently. Symptoms associated with uncomplicated UTI caused by Gram-positive uropathogens are similar to those caused by Gram-negative organisms. Complicated UTIs often occur in nosocomial settings, particularly in individuals with structural or functional alterations of the urinary tract (often associated with urinary catheterization), or other underlying renal, metabolic, or immunological disorders [22]; those populations are at greater risk of Gram-positive and polymicrobial UTI of [23]. In spite of being usually seen in small numbers of cultured urine samples, Staphylococcus aureus, coagulase negative Staphylococcus, Streptococci and Enterococci are recognized as important causes of UTI [24,25].

In this study, we investigated the frequency and antimicrobial susceptibility patterns of Gram-positive cocci isolated from patients with nosocomial UTI at the maternity and child hospital (MHC) in Medina, KSA.

In this study, 14.5% of our urine isolates were gram-positive cocci (165/1137) (Figure 1). Higher isolation rates of gram-positive cocci from urine were reported in other studies [18,21,26]. The variations in the type and distribution of UTI-causing bacteria in various geographic regions and countries may be due to training, host conditions, hygiene practices, and healthcare practices.

Enterococcus faecalis isolated from children were 62 out of 109 (56.9 %) and 26 out of 56 in adult females (46.4%) while Staphylococcus aureus was the least isolated organism in children (3/109) (2.7%) and Enterococcus faecium was the least isolated organism among adult females (1/56) (1.8%) (Figure 2). This result is different from that of Tayebi et al, 2014 [27] who reported that S. aureus was the most common gram-positive cocci isolated in both sexes and all age-groups (S. aureus strains were responsible for 59.8% of UTI cases).

The incidence of UTI varies according to the age and sex of children. It is known that UTI is more frequent in boys in the first 2 years of life (period of infancy). In the present study this was also true. Gram positive cocci were isolated from 28 infants ≤ 2 years and percentage of gram positive UTIs was significantly higher in male (71.4% “20/28”) than female (28.6% “8/28”) infants (Figure 3). This was very close to the value arrived at in the study done by Saeed and coworkers, 2015 [28] who reported that the percentage of UTIs was significantly higher in males (60.9%) than females (39.1%) during the first 3 months of life. This male to female preponderance during infancy, may be due to congenital obstruction, phimosis and high frequency of urethral malformations in male infants [29], In addition to high frequency of UTI in uncircumcised boys [30]. Another study [31] found that there are almost equal numbers of cases of UTI for the first two years of life in male and female patients, and then a marginally increasing rate of UTI was found among female children older than two years.

Among children with isolated gram-positive cocci from urine, only one third of cases (37.6%) were presented with urinary tract infections while most cases (68/109) (62.3%) were presented with other conditions including chest infections, polycystic kidney, hydronephrosis, sepsis, epilepsy, colitis and sickle sick anaemia (Figure 4). Multiple studies mentioned that the classical features of UTI are absent in young children, who often present with few signs or symptoms other than fever [32,33,34]. Urinalysis in febrile babies, especially boys younger than 12 months, is important to consider because febrile urinary tract infections (UTI) have long been considered amongst the most common serious bacterial infections in childhood with renal scarring a frequent outcome [35]. The majority of paediatric cases (55%) in the current study were admitted in inpatient paediatric departments, while 36.7% were from paediatric emergency department and 6.4% in neonatal intensive care units. Minority of cases were from other departments as nephrodialysis and behaviour and growth units (Figure 5).

The rate of UTIs gradually increases with age to 30 years in women (average 20-40 years which is the childbearing period) [36]. Regarding adult females in our study, 42 out of 56 (75%) presented with urinary tract infection associated with pregnancy while the other 14 (25%) presented with UTI associated with other conditions such as vaginitis, abnormal vaginal bleeding and fever (Figure 6). In addition, we had a 34% (19/56) prevalence of asymptomatic bacteriuria in our study population. Our value is much higher than the range of 2-10% reported elsewhere [37,38,39]. Variation in studies may be due to differences in geographical location, socioeconomic status, setting of study (primary care, general hospital and community), sample size and variation in screening tests (cut-off point for the detection of pathogens).

For long time, enterococci were frequently considered to be commensal organism and was ignored when isolated in clinical laboratory. But recently its capability of causing variety of infections, especially in hospitalized patients, and difference in antimicrobial sensitivity of each species to varying antibiotics have led to understanding the importance of identification of Enterococcus to species level. Enterococcus species were by far the most common gram-positive cocci isolated from urine samples in our study (71.9%). E. faecalis was the predominant species (53.3% of isolates) (Table 2), which is not surprising, in light of global epidemiological reports on the causative agents for UTIs [10] while was contrary to the findings of others [40,41] who reported Staphylococcus species as the most commonly isolated gram positive uropathogen. The high incidence of enterococcal UTI may be attributed to the underlying condition, longer hospital stay, colonization of Foley's catheters and increasing use of broad-spectrum antibiotics particularly cephalosporins as the case in our hospital.

The increased incidence of enterococcal UTI is alarming; resistance to most commonly used antimicrobial agents is a typical characteristic of these bacteria. In the current study, 75% of Enterococcus faecalis and 50% of Enterococcus faecium isolates were sensitive to nitrofurantoin (Figure 9). This result was comparable with that published by Al-Tawfiq and Anani, 2009 [42] who found that the susceptibility of HCA and CA isolates of E. faecalis to nitrofurantoin were 78.3 and 93.6%, respectively. Several other investigators have reported isolates that have reduced sensitivity to nitrofurantoin [43]. 18% of Enterococcus faecalis isolates were sensitive to gentamycin. Gajdács et al, 2020 reported that high-level aminoglycoside resistance in enterococci was noted in 31.0-46.6% of cases [44].

Data from our investigation showed varying susceptibility to antibiotics with significant resistance of Enterococcus spp isolates (more than 80%) to erythromycin, ampicillin, cefazolin, ceftriaxone, norfloxacin, and sulfamethoxazole-trimethoprim. The motivation for resistance to these antibiotics could be their abuse in the hospitals and the community. All the enterococcal isolates (other than VRE isolates) were susceptible to vancomycin and linezolid. This finding is similar to that published in the study done by Kaur et al., 2014 in India [45].

S. aureus is an important uropathogen and was responsible for 10.7% (6/56) of UTI cases among adult females in our study (Figure 2). This result matched with that of Tchente Nguefack et al, 2019 who reported that S. aureus accounted for 8.6% of pathogens involved in bacteriuria of pregnant women [46]. Similarly, Baraboutis et al, 2010 stated that in contrast to S. saprophyticus, which is a predominant cause of community-acquired UTI, S. aureus UTI more often occurs in urinary-catheterized and pregnant individuals [47]. It has been emphasized that any amount of this bacterium should be subjected to antibiogram test [48].

Over the last decade there has been a substantial increase in resistance of S. aureus strains to antibiotics. In this study, fortunately resistant strains to cefazolin, nirtofurantoin, sulfamethoxazole and vancomycin were not observed (Figure 9). Similar findings were present in the study of Tayebi et al, 2014 [41]. Based on our study, a high level of resistance to ampicillin was seen among Staphylococcus aureus isolates. In concordance with other studies, these results implied that ampicillin cannot be used for treatment of UTI [49,50].

Streptococcus agalactiae, otherwise known as group B Streptococcus (GBS) is a gram-positive β-hemolytic chain-forming coccus that is a common asymptomatic inhabitant of the lower gastrointestinal and female reproductive tracts. In our study, GBS was estimated to cause 3.4% (39/1137) of all monomicrobial UTIs (Table 2). Our result approached to that reported by Foxman, 2003 [51]. Asymptomatic bacteriuria (ASB) caused by GBS is common among pregnant females with higher risk of ascending pyelonephritis that can progress to bacteremia and/or urosepsis [52].

In addition, GBS is the leading cause of sepsis and meningitis in newborns and can be acquired by the newborn in utero or during passage through the colonized birth canal [53]. This was apparent in our study in which GBS were isolated from 9 out of 19 cases (47.4%) of asymptomatic bacteriuria among pregnant females included in the study.

All isolates of Streptococcus agalactiae in our study were sensitive to cephalocin and nitrofurantoin, while 52% of them were resistant to ampicillin and all were resistant to Trimethoprim-sulfamethoxazole (Figure 6). This result was matched with that arrived by Alhambra et al, 2004 [54] who found that S. agalactiae isolates were susceptible to the β-lactams and nitrofurantoin used in the treatment of UTIs caused by this species. However, levels of resistance to macrolides and lincosamides were significant (20%) in their study and most isolates (83.5%) were resistant to tetracyclines.

(MDR) was defined as resistance to at least three or more antibiotics [12]. In the current study, multidrug resistant positive cocci formed 9.7% (16/165) of total gram-positive isolates (Figure 7) including vancomycin resistant enterococci (VRE) (8.5%) and methicillin resistant staph aureus (MRSA) (1.2%) (Figure 8). Recent studies have reported the increasing prevalence of multi drug resistant S. aureus especially MRSA in UTIs [55] Tayebi et al, 2014 reported of 316 isolates tested 151 (47.8%) were MDR [27]. All the 16 MDR stains were isolated from infants (15 0f them were less than 2 years); 10 males and 6 females and all were admitted in the hospital. It could be attributable to high usage of antimicrobial agents in the hospitals. Monitoring of antimicrobial susceptibility helps prevent the spread of resistant isolates and eliminate the use of antibiotics for a prolonged period [56,57].

Only one third of our MDR-infected infants presented with symptoms of UTI (31.2%) and the mortality rate among them was 6.3% (1/16) (Table 3). VRE colonization is seen more frequently in newborns who have medical problems during follow-up. Therefore, surveillance cultures that performed routinely in NICUs, would be helpful to detect VRE colonization in time and to implement isolation precautions rapidly to prevent dissemination of the organism and decrease the incidence of bacteremia and death [58].

Among the strains of Enterococcus spp. investigated in this study, 14/117 (11.9%) were VRE. The 14 VRE isolates were Enterococcus faecium. This result matched the study of Gajdács et al, 2020 in which all urinary VRE isolates were exclusively from E. faecium [44]. Among E. faecium isolates, vanB genotype was detected. The predominance of E. faecium vanB was similarly reported in an Australian paper [59] and suggested an epidemiology different from that in either Europe or the United States. On the other hand, a study held in a tertiary hospital in Saudi Arabia and reported that vanA+/vanB+ with vanB phenotype were predominant [60]. Another study conducted in King Faisal Hospital in Rhiyad showed predominant vanA genotype and provided the first report of vanB phenotype- vanA genotype incongruent E. faecium in the Middle East region [61]. Molecular typing indicates clonal spread and high occurrence of virulence genes associated with epidemic clones.

5. Conclusion

Gram-positive organisms recovered from urine are found more often with increasing levels of antibiotic resistance. Continuous Surveillance for multidrug-resistant strains is necessary to prevent the further spread of resistant isolates and to provide physicians with knowledge about the most effective empirical treatment of UTIs. From the present study, vancomycin and nitrofurantoin seem to be effective drugs for treatment of UTI associated with gram positive cocci. According to our findings, Ampicilin, Ceftriaxone, erythromycin and Sulfamethoxazole-trimethoprim are not effective drugs for treatment of gram positive UTI. Molecular typing of VRE strain in this study revealed vanB genotype.

Acknowledgements

The authors would like to thank the staff of Maternity and child hospital in Medina for their assistance in this study.

Ethics Approval and Consent to Participate

1. The study protocol was approved by the Studies and Research Committee, MMCH, General Directorate of Health, Madinah, Saudi Arabia.

2. Maintaining confidentiality of information obtained from subjects under the study.

3. Results of samples collected were donated to physicians of patients included in the study for treatment prescription.

Conflict of Interest

The authors declare that they have no conflict of interests.

References

[1]   | Barišić Z, Babić-Erceg A, Borzić E, Zoranić V, Kaliterna V, Carev M. Urinary tract infections in South Croatia: aetiology and antimicrobial resistance. Int J Antimicrob Agents. 2003; 22: 61-4.
[2]   | Prais D, Straussberg R, Avitzur Y, Nussinovitch M, Harel L, Amir J. Bacterial susceptibility to oral antibiotics in community acquired urinary tract infection. Arch Dis Child. 2003; 88(3): 215-8.
[3]   | Foxman B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Am J Med. 2002; 113(1): 53-70.
[4]   | Mazzulli T. Resistance trends in urinary tract pathogens and impact on management. J Urol. 2002; 168(4 Part 2): 1720-2.
[5]   | Maki DG, Tambyah PA. Engineering out the risk for infection with urinary catheters. Emerg Infect Dis. 2001; 7(2): 342.
[6]   | Finer G, Landau D. Pathogenesis of urinary tract infections with normal female anatomy. Lancet Infect Dis. 2004; 4(10): 631-5.
[7]   | Brook I. Urinary tract and genito‐urinary suppurative infections due to anaerobic bacteria. Int J Urol. 2004; 11(3): 133-41.
[8]   | Le J, Briggs GG, McKeown A, Bustillo G. Urinary tract infections during pregnancy. Ann Pharmacother. 2004; 38(10): 1692-701.
[9]   | Sefton AM. The impact of resistance on the management of urinary tract infections. Int J Antimicrob Agents. 2000; 16(4): 489-91.
[10]   | Hryniewicz K, Szczypa K, Sulikowska A, Jankowski K, Betlejewska K, Hryniewicz W. Antibiotic susceptibility of bacterial strains isolated from urinary tract infections in Poland. J Antimicrob Chemother. 2001; 47(6): 773-80.
[11]   | Bonadio M, Meini M, Spitaleri P, Gigli C. Current microbiological and clinical aspects of urinary tract infections. Eur Urol. 2001; 40(4): 439-45.
[12]   | Traub WH, Leonhard B. Agar disk diffusion (Bauer-Kirby) tests with various fastidious and nonfastidious reference (ATCC) strains: Comparison of several agar media. Chemotherapy. 1994; 40(6): 374-83.
[13]   | Weinstein MP, Lewis JS. The Clinical and Laboratory Standards Institute Subcommittee on Antimicrobial Susceptibility Testing: Background, Organization, Functions, and Processes. J Clin Microbiol. 2020; 58(3).
[14]   | Bell JM, Paton JC, Turnidge J. Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates. J Clin Microbiol. 1998; 36(8): 2187-90.
[15]   | Perichon B, Reynolds P, Courvalin P. VanD-type glycopeptide-resistant Enterococcus faecium BM4339. Antimicrob Agents Chemother. 1997; 41(9): 2016-8.
[16]   | Biosciences A. Isolation and antimicrobial susceptibility testing of Escherichia coli causing urinary tract infections. October. 2009; 22: 1320-5.
[17]   | Assefa A, Asrat D, Woldeamanuel Y, Abdella A, Melesse T. Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at Tikur Anbessa Specialized Hospital Addis Ababa, Ethiopia. Ethiop Med J. 2008; 46(3): 227-35.
[18]   | Farajnia S, Alikhani MY, Ghotaslou R, Naghili B, Nakhlband A. Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran. Int J Infect Dis. 2009; 13(2): 140-4.
[19]   | Balkhy HH, Cunningham G, Chew FK, Francis C, Al Nakhli DJ, Almuneef MA, et al. Hospital-and community-acquired infections: a point prevalence and risk factors survey in a tertiary care center in Saudi Arabia. Int J Infect Dis. 2006; 10(4): 326-33.
[20]   | Angoti G, Goudarzi H, Hajizadeh M, Tabatabaii Z. Bacteria isolated from urinary tract infection among patients and determination of the antibiotic susceptibility patterns of the gram negative bacteria in Iran. Nov Biomed. 2016; 4(1): 1-4.
[21]   | Raka L, Mulliqi-Osmani G, Berisha L, Begolli L, Omeragiq S, Parsons L, et al. Etiology and susceptibility of urinary tract isolates in Kosova. Int J Antimicrob Agents. 2004; 23: 2-5.
[22]   | Wagenlehner FME, Naber KG. Current challenges in the treatment of complicated urinary tract infections and prostatitis. Clin Microbiol Infect. 2006; 12: 67-80.
[23]   | Matthews SJ, Lancaster JW. Urinary tract infections in the elderly population. Am J Geriatr Pharmacother. 2011; 9(5): 286-309.
[24]   | Esmaeili M. Antibiotics for causative microorganisms of urinary tract infections. 2005;
[25]   | Mohammad-Jafari H, Saffar MJ, Nemate I, Khalilian HSA-R. Increasing antibiotic resistance among uropathogens isolated during years 2006-2009: impact on the empirical management. Int braz j urol. 2012; 38(1): 25-32.
[26]   | Tessema B, Kassu A, Mulu A, Yismaw G. Pridominant isolates of urinary tract pathogens and their antimicrobial susceptiblity patterns in Gondar University Teaching Hospital, nothwest Ethiopia. Ethiop Med J. 2007; 45(1): 61.
[27]   | Tayebi Z, Seyedjavadi SS, Goudarzi M, Rahimi MK, Boromandi S, Bostanabad SZ, et al. Frequency and antibiotic resistance pattern in gram positive uropathogenes isolated from hospitalized patients with urinary tract infection in Tehran. Iran J Genes Microb Immun. 2014; 2014: 1-9.
[28]   | Mansoor IY, AL-Otraqchi KIB, Saeed CH. Prevalence of urinary tract infections and antibiotics susceptibility pattern among infants and young children in Erbil city. Zanco J Med Sci. 2015; 19(1): 915-22.
[29]   | Kanellopoulos TA, Salakos C, Spiliopoulou I, Ellina A, Nikolakopoulou NM, Papanastasiou DA. First urinary tract infection in neonates, infants and young children: a comparative study. Pediatr Nephrol. 2006; 21(8): 1131-7.
[30]   | Yilmaz Y, Tazegun ZT, Aydin E, Dulger M. Bacterial uropathogens causing urinary tract infection and their resistance patterns among children in Turkey. Iran Red Crescent Med J. 2016; 18(6).
[31]   | Shaikh N, Morone NE, Bost JE, Farrell MH. Prevalence of urinary tract infection in childhood: a meta-analysis. Pediatr Infect Dis J. 2008; 27(4): 302-8.
[32]   | Sivaraj P, Pradhan S, Beriha S, Barik R, Tago N, Das L, et al. Study of risk factors associated with febrile urinary tract infection in children. Sri Lanka J Child Heal. 2015; 44(3).
[33]   | Korbel L, Howell M, Spencer JD. The clinical diagnosis and management of urinary tract infections in children and adolescents. Paediatr Int Child Health. 2017; 37(4): 273-9.
[34]   | Shrestha LB, Baral R, Poudel P, Khanal B. Clinical, etiological and antimicrobial susceptibility profile of pediatric urinary tract infections in a tertiary care hospital of Nepal. BMC Pediatr. 2019; 19(1): 36.
[35]   | Sakran W, Miron D, Halevy R, Colodner R, Smolkin V, Koren A. Community acquired urinary tract infection among hospitalized children in northern Israel: pathogens, susceptibility patterns and urinary tract anomalies. Harefuah. 2003; 142(4): 249-52.
[36]   | Glover M, Moreira CG, Sperandio V, Zimmern P. Recurrent urinary tract infections in healthy and nonpregnant women. Urol Sci. 2014; 25(1): 1-8.
[37]   | Easter SR, Cantonwine DE, Zera CA, Lim K-H, Parry SI, McElrath TF. Urinary tract infection during pregnancy, angiogenic factor profiles, and risk of preeclampsia. Am J Obstet Gynecol. 2016; 214(3): 387-e1.
[38]   | Marzieh J, Mohsen S, Nasrin R, Koroosh K. Prevalence of urinary tract infection and somes factors affected in pregnant women in Iran, Karaj. Middle-East J Sci Res. 2014; 20(7): 782-5.
[39]   | Labi AK, Yawson AE, Ganyaglo GY, Newman MJ. Prevalence and associated risk factors of asymptomatic bacteriuria in ante-natal clients in a large teaching hospital in Ghana. Ghana Med J. 2015; 49(3): 154-8.
[40]   | Khoshbakht R, Salimi A, SHIRZAD AH, Keshavarzi H. Antibiotic susceptibility of bacterial strains isolated from urinary tract infections in Karaj, Iran. 2013;
[41]   | von Eiff C, Proctor RA, Peters G. Coagulase-negative staphylococci: pathogens have major role in nosocomial infections. Postgrad Med. 2001; 110(4): 63-76.
[42]   | Al-Tawfiq JA, Anani AA. Antimicrobial susceptibility pattern of bacterial pathogens causing urinary tract infections in a Saudi Arabian hospital. Chemotherapy. 2009; 55(2): 127-31.
[43]   | Sheikholeslami NZ, Hassanshahi G. The frequency of coagulase negative staphylococci urinary infections with antimicrobial resistance pattern in Rafsanjan. Pakistan J Med Sci. 2010; 26(1): 107-10.
[44]   | Gajdács M, Ábrók M, Lázár A, Burián K. Increasing relevance of Gram-positive cocci in urinary tract infections: A 10-year analysis of their prevalence and resistance trends. Sci Rep. 2020; 10(1): 1-11.
[45]   | Kaur N, Sharma S, Malhotra S, Madan P, Hans C. Urinary tract infection: aetiology and antimicrobial resistance pattern in infants from a tertiary care hospital in northern India. J Clin diagnostic Res JCDR. 2014; 8(10): DC01.

[46]  
Nguefack CT, Ebongue CO, Chokotheu CN, Ewougo CE, Njamen TN, Mboudou E. Clinical presentation, risk factors and pathogens involved in bacteriuria of pregnant women attending antenatal clinic of 3 hospitals in a developing country: a cross sectional analytic study. BMC Pregnancy Childbirth. 2019; 19(1): 143.

[47]   | Baraboutis IG, Tsagalou EP, Lepinski JL, Papakonstantinou I, Papastamopoulos V, Skoutelis AT, et al. Primary Staphylococcus aureus urinary tract infection: the role of undetected hematogenous seeding of the urinary tract. Eur J Clin Microbiol Infect Dis. 2010; 29(9): 1095-101.
[48]   | Al-Ali SM, Al-Faraj JM, Al-Muslim SS. Antimicrobial resistance pattern in urinary tract pathogens and its impact on empirical therapy in general practice. Kuwait Med J. 2005; 37: 22-7.
[49]   | Haller M, Brandis M, Berner R. Antibiotic resistance of urinary tract pathogens and rationale for empirical intravenous therapy. Pediatr Nephrol. 2004; 19(9): 982-6.
[50]   | Sharifian M, Karimi A, Tabatabaei SR, Anvaripour N. Microbial sensitivity pattern in urinary tract infections in children: a single center experience of 1,177 urine cultures. Jpn J Infect Dis. 2006; 59(6): 380.
[51]   | Foxman B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Disease-a-month. 2003; 49(2): 53-70.
[52]   | Ulett KB, Benjamin WH, Zhuo F, Xiao M, Kong F, Gilbert GL, et al. Diversity of group B streptococcus serotypes causing urinary tract infection in adults. J Clin Microbiol. 2009; 47(7): 2055-60.
[53]   | Kessous R, Weintraub AY, Sergienko R, Lazer T, Press F, Wiznitzer A, et al. Bacteruria with group-B streptococcus: is it a risk factor for adverse pregnancy outcomes? J Matern Neonatal Med. 2012; 25(10): 1983-6.
[54]   | MIC90 RM. isolates from blood and urine to 18 widely used and recently marketed antibiotics. Clin Microbiol Infect. 2004; 10(3).
[55]   | Looney AT, Redmond EJ, Davey NM, Daly PJ, Troy C, Carey BF, et al. Methicillin-resistant Staphylococcus aureus as a uropathogen in an Irish setting. Medicine (Baltimore). 2017; 96(14).
[56]   | Mansour A, MAHDINEZHAD M, Pourdangchi Z. Study of bacteria isolated from urinary tract infections and determination of their susceptibility to antibiotics. 2009;
[57]   | Goudarzi M, Seyedjavadi SS, Goudarzi H, Boromandi S, Ghazi M, Azad M, et al. Characterization of coagulase-negative staphylococci isolated from hospitalized patients in Tehran, Iran. J Paramed Sci Spring. 2014; 5(2): 2008-4978.
[58]   | Benzer D, Yavuzcan ÖD, Gürsoy T, Ocalmaz MS, Karatekin G, Ovalı HF. Vancomycin-resistant enterococcus colonization in neonatal intensive care unit: prevention and eradication experience. Mikrobiyol Bul. 2012; 46(4): 682.
[59]   | Leclercq R, Courvalin P. Resistance to glycopeptides in enterococci. Clin Infect Dis. 1997; 24(4): 545-54.
[60]   | Somily AM, Al-Mohizea MM, Absar MM, Fatani AJ, Ridha AM, Al-Ahdal MN, et al. Molecular epidemiology of vancomycin resistant enterococci in a tertiary care hospital in Saudi Arabia. Microb Pathog. 2016; 97: 79-83.
[61]   | Al-Ahdal MN, Abozaid SM, Al-Shammary HF, Bohol MF, Al-Thawadi SI, Al-Jaberi AA, et al. Characterization of Enterococcus faecium isolates and first report of vanB phenotype-vanA genotype incongruence in the Middle East. Eur J Clin Microbiol Infect Dis. 2012; 31(11): 3223-9.