Routine Bacteriological Methods Research Articles

Relationship of biofilm formation and different virulence genes in uropathogenic Escherichia coli isolates from Northwest Iran.

Background and objectives: The Escherichia coli (E. coli) bacterium is one of the main causative agents of urinary tract infections (UTI) worldwide. The ability of this bacterium to form biofilms on medical devices such as catheters plays an important role in the development of UTI. The aim of the present study was to investigate the possible relationship between virulence factors and biofilm formation of E. coli isolates responsible for urinary tract infection.Materials and methods: A total of 100 E. coli isolates isolated from patients with UTI were collected and characterized by routine bacteriological methods. In vitro biofilm formation by these isolates was determined using the 96-well microtiter-plate test, and the presence of fimA, papC, and hly virulence genes was examined by PCR assay. Data analysis was performed using SPSS 16.0 software.Results: From 100 E. coli isolates isolated from UTIs, 92% were shown to be biofilm positive. The genes papC, fimA, and hly were detected in 43%, 94% and 26% of isolates, respectively. Biofilm formation in isolates that expressed papC, fimA, and hly genes was 100%, 93%, and 100%, respectively. A significant relationship was found between presence of the papC gene and biofilm formation in E. coli isolates isolated from UTI (P<0.01), but there was no statistically significant correlation between presence of fimA and hly genes with biofilm formation (P<0.072, P<0.104). Conclusion: Results showed that fimA and hly genes do not seem to be necessary or sufficient for the production of biofilm in E. coli, but the presence of papC correlates with increased biofilm formation of urinary tract isolates. Overall, the presence of fimA, papC, and hly virulence genes coincides with in vitro biofilm formation in uropathogenic E. coli isolates.

Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique

Bacterial L-forms have always been considered as osmotic-pressure-sensitive cell-wall-deficient bacteria and isolation culture of L-forms must use media with high osmotic pressure. However, isolation culture of stable L-forms formed in humans and animals is very difficult because they have adapted to the physiological osmotic pressure condition of the host. We use a non-high osmotic isolation technique to isolate stable L-forms of Salmonella Typhi and Salmonella Paratyphi A from bile-inducer cultures in vitro and from patients' gallbladder specimens. Multiplex PCR assay for Salmonella-specific genes and nucleotide sequencing are used to identify the Salmonella L-forms in stable L-form isolates. Using this method, we confirmed that Salmonella Paratyphi A and Salmonella Typhi cannot be isolated from bile-inducer cultures cultured for 6 h or 48 h, but the L-forms can be isolated from 1 h to 45 days. In the 524 gallbladder samples, the positive rate for bacterial forms was 19.7% and the positive rate for Salmonella spp. was 0.6% by routine bacteriological methods. The positive rate for bacterial L-forms was 75.4% using non-high osmotic isolation culture. In the L-form isolates, the positive rate of Salmonella invA gene was 3.1%. In these invA-positive L-form isolates, four were positive for the invA and flic-d genes of Salmonella Typhi, and ten were positive for the invA and flic-a genes of Salmonella Paratyphi A.

Identification of blood and wound isolates of C. canimorsus and C. cynodegmi using VITEK2 and MALDI-TOF

Capnocytophaga canimorsus and C. cynodegmi are gram negative bacteria that can be transmitted to humans from dogs or cats and cause serious infections. Routine bacteriological methods, including fermentation and phenotypic tests are insufficient to correctly identify C. canimorsus or C. cynodegmi. The aim of this study was to evaluate the performance of VITEK2 and MALDI-TOF in identification of these bacteria. Twenty two isolates that were identified as C. canimorsus / C. cynodegmi by 16S rRNA sequencing were included in the study and were further investigated with VITEK2 and MALDI-TOF. A Capnocytophaga species-specific PCR was used as the reference method. Out of 22 included isolates, the species-specific PCR identified six blood isolates as C. canimorsus and 14 wound isolates as C. cynodegmi. Two isolates could not be identified with the reference method. VITEK2 identified 10/20 isolates correctly to Capnocytophaga spp. MALDI-TOF analysis correctly identified 6/6 C. canimorsus and 13/14 C. cynodegmi isolates. The mean time to identification with VITEK2 was 6 hours whereas MALDI-TOF required approximately 10 minutes per sample. Here we show that MALDI-TOF rapidly identified C. canimorsus and C. cynodegmi and thus constitutes a valuable diagnostic tool in the clinical laboratory.

Hematoma and abscess formation caused by Mycoplasma hominis following cesarean section

Mycoplasma species cannot be identified by routine bacteriological culture methods and are resistant to common antimicrobial agents. Mycoplasma hominis usually colonizes the lower urogenital tract and causes pyelonephritis, pelvic inflammatory disease, chorioamnionitis, rupture of fetal membranes, preterm labor, postpartum fever, postabortal fever, and neonatal infection. This organism is highly prevalent in cervicovaginal cultures of sexually active women. M. hominis, M. genitalis, Ureaplasma urealyticum, and U. parvum may invade and infect placental and fetal tissues, leading to adverse pregnancy outcomes. M. hominis occasionally causes nongenitourinary infection of the blood, wounds, central nervous system, joints, or respiratory tract. We present a case of a 27-year-old woman who developed abdominal wound hematoma and abscess after cesarean section. The wound was drained, but her high fever persisted, in spite of antibiotic treatment using flomoxef sodium and imipenem·cilastatin sodium. Because the exudate exhibited M. hominis growth in an anaerobic environment, we administered the quinolone ciprofloxacin. This therapy resolved her fever, and her white blood cell count and C-reactive protein level diminished to the normal ranges. To our knowledge, there are four published articles regarding the isolation of M. hominis from postcesarean incisions. Based on the current study and the literature, infection by this pathogen may cause hematoma formation with or without abscess after cesarean section or in immunosuppressed postoperative patients. In such cases, physicians may need to suspect Mycoplasma infection and initiate appropriate antibacterial treatment as soon as possible in order to avoid persistent fever.

An outbreak of shigellosis due to Shigella flexneri serotype 3a in a prison in Iran.

On June 16 and 17, 2007, the medical clinic of a prison in Isfahan, Iran received multiple reports of gastrointestinal illness among prisoners. A cross-sectional study was therefore undertaken to determine the extent, causative agent and possible source of the outbreak. A case-patient was defined and patient information was collected with a standardized questionnaire. Stool samples were collected from the patients and restaurant employees, and analyzed for the presence of enteric bacteria by routine bacteriological methods. Shigella isolates were identified and serotyped by commercially available antisera. The relationship between the strains was determined using antimicrobial drug resistance pattern analysis and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Seven hundred one inmates experienced gastrointestinal illness and severe diarrhea. The attack rate was 14.02%. Rectal swabs and stool cultures recovered from patients tested positive for Shigella flexneri serotype 3a. All tested isolates had a similar antibiotic resistance and ERIC-PCR pattern. Our findings demonstrated that raw vegetables were more likely to be the causative agent of this outbreak. The results indicated that a single clone of S. flexneri was responsible for this outbreak. Although we could not trace the exact origin of the organism, the consumption of raw vegetables one day prior to the onset of illness was strongly associated with an increased risk of S. flexneri infection. This study emphasizes the need for accurate monitoring and surveillance of food and vegetables consumed in prisons.

Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients

Background Longitudinal data regarding the genotypes of Pseudomonas aeruginosa isolates after eradication treatment are limited. We followed cystic fibrosis patients after a first ever isolation of P. aeruginosa and evaluated the P. aeruginosa-free time period after eradication therapy. Methods Between January 2003 and December 2008 respiratory samples were cultured prospectively from 41 patients with a first ever P. aeruginosa isolate. Twenty five patients had at least one subsequent isolate. Treatment efficacy was assessed based on the time to a second isolation and on comparison of the RAPD genotypes of the P. aeruginosa isolates. Results Eleven patients became chronically colonized during the study period. For ten of these the second isolate had the same genotype as the first isolate. Moreover, these patients had a significantly shorter P. aeruginosa-free time interval between the first ever and the second isolate compared to the 14 not chronically colonized patients (median 0 months versus 7.5 months, p < 0.05). Conclusion Our results indicate that the presence of a genotypically identical subsequent P. aeruginosa isolate and/or a short P. aeruginosa-free time interval after treatment are ominous signs and might be useful additional tools to predict impending chronic colonization. Current routine bacteriological methods for the detection of P. aeruginosa may lack the sensitivity to discriminate between true eradication and low bacterial persistence.

An evaluation of the hand and nasal flora of Turkish nursing students after clinical practice

The purpose of this study was to evaluate and compare the hand and nasal flora of nursing students before and after the clinical practice. Hospitals are places where infective agents abound. Healthcare workers, relatives of patients and students practising in the hospital medium are often exposed to these infective agents. Although the role of the hand and nasal flora of healthcare workers in the development of nosocomial infections has been emphasised by earlier studies, there are a limited number of studies which investigate the hand and nasal flora of nursing students. Descriptive. This descriptive study involved 66 volunteer nursing students. Two samples of flora from both hands and nose of each student were obtained. The inoculated samples were then evaluated through routine bacteriological study methods. Chi-square and percentage calculations were used in comparisons. None of the students had methicillin-resistant Staphylococcus aureus or methicillin-resistant coagulase-negative Staphylococcus colonisation in the hand samples before clinical practice, 6.1% of the students had methicillin-resistant Staphylococcus aureus and 4.5% had methicillin-resistant coagulase-negative Staphylococcus colonisation after the practice. Although the differences between the rates of contamination with pathogen micro-organisms in the hand and nasal flora of the student nurses before and after clinical practice were not significant, the rate of colonisation after clinical practice was higher. In this study, the rate of colonisation after clinical practice was higher. These findings indicate that students might have been contaminated with bacteria during clinical practice. The results of this study have practical importance in clinical practice. The role of the hand and nasal flora of nursing students in the development of nosocomial infections is significant. For this reason, some precautions, such as using gloves and handwashing with special solutions when needed, should be taken to prevent nosocomial infections and protect students against associated risks.

Ability of Iranian Microbiology Laboratories for Detection of Methicillin Resistant Staphylococcus aureus and Performance of Susceptibility to Vancomycin

an Iranian 1000-bed hospital and to assess the activity of commonly used antibiotics including nitrofuantoin against VRE isolates. Methods: This study was carried out between April 2002 and April 2006 in Milad Hospital of Tehran a 1000-bed nonteaching hospital Only patients who had puria and significant bactteriuria included in our study. All isolates performed identification to species level by using routine bacteriological methods and susceptibility testing performed by disk diffusion method as recommended by Clinical Laboratory Institute Standards (CLSI) Results: All urine isolates were also tested for resistance to ampicillin, penicillin, ciprofloxacin, erythromycin, tetracycline and norfloxacine. Results: In Total 837 Enterococcus species were isolated from UTI that 668 (79.8%) and 169 (20.2%) isolates were E.faecalis and E.faecium respectively. Analysis of the collected data revealed that 9.43% vancomycin resistant strains were resistant to nitrofurantoin as well (6 out of 65), while 84.9% of the isolated entrococci (54 out 65) were vancomycin resistant and nitrofurantoin sensitive resistance among E.faecium were prevalent in comparison with E. feaclis isolates Study on the data also showed both vancomycin and nitrofurantoin resistant isolate have higher frequency rate in admitted patients compared with out-patients. Conclusion: It is concluded that susceptibility approximately 85% VRE to nitroforantoin still is the suitable alternative drug for treatment urinary tract infections caused by VRE.

Ribotyping of Salmonella Enteritidis strains reveals the spread of a single genotype in the Brazilian city of Ribeirão Preto

BACKGROUND: The approach generally employed in leading laboratories worldwide to identify strains of Salmonella is to detect epidemiological markers by serotyping and molecular techniques. These molecular methods are important in sanitary surveillance and lead to the source of infection. OBJECTIVES: The aim of the present study is to characterize Salmonella Enteritidis strains by ribotyping. MATERIAL AND METHODS: Thirty-eight strains of S. Enteritidis were isolated from in patients at the university hospital of Universidade de São Paulo, Ribeirão Preto, SP, Brazil, between 1996 and 1998. These strains were isolated from stools (31 samples), blood (4 samples) and other body fluids (3 samples), using routine bacteriological methods, and were serotyped and ribotyped. RESULTS: The 38 strains of serotype S. Enteritidis were shown by the ribotyping to belong to two ribotypes: A (94.7% of the samples) and B (5.3%). DISCUSSION AND CONCLUSION: These results suggest that the majority of patients (94.7%) were infected by the same strain. This strain could be endemic in the Ribeirão Preto community, or these patients may have been exposed to a common source of infection.

Molecular subtyping of Campylobacter jejuni subsp. jejuni strains isolated from different animal species in the state of São Paulo, Brazil

The objective of the present trial was to characterize genetically strains of Campylobacter jejuni subsp. jejuni isolated from humans and several animal sources (bovines, swine, dogs, primates, wild boars and poultry). A total of 828 different animal samples (feces, carcass, aborted fetus and hysterectomized uterus) were analysed by means of routine bacteriological methods, and 36 C. jejuni strains were isolated. Thirty strains of human fecal origin were obtained in clinical analysis laboratories in the city of Sao Paulo. The 66 C. jejuni strains isolated were submitted to genetic characterization. Primers based on fla A gene were used in a polymerase chain reaction (PCR) and amplified a fragment of the 702 bp. PCR products were evaluated by means of sequencing and genealogic analysis. Genetic variability analysis of 66 strains showed 44 different subtypes of C. jejuni. One subtype was identical to a C. jejuni strain of human origin with the sequence in the GenBank (GENBANK accession number AF050186). Subtyping analysis of C. jejuni strains based on sequencing of the fla A gene variable region and analysis of sequence alignment by the Maximum Parsimony method showed to be highly discriminatory, providing the best conditions to differentiate strains involved in outbreaks from those sporadically isolated. This is the first study of molecular subtyping analysis of human and animal C. jejuni strains using sequencing technique and genealogic analysis in the state of Sao Paulo, Brazil.

Evaluation of diagnostic procedures for subclinical mastitis in meat-producing sheep.

Samples of foremilk were collected from 261 clinically normal glands of 150 ewes, and tested using the California mastitis test (CMT). Further samples were collected from 195 of these glands for determination of automated somatic cell counts (SCC), and from 60 of these glands for bacteriological assessment. The sensitivity and specificity of CMT for detecting samples with SCC above different threshold levels and for CMT and SCC in determining bacteriological status were evaluated using two-graph receiver operating characteristics (TG-ROC). Milk samples were obtained subsequently from ten CMT positive, and five CMT negative first- and second-lactation ewes. Samples were cultured using a variety of media, incubation temperatures and atmospheric conditions, immediately after collection, and 1 week after storage at 4 degrees C and -21 degrees C. Results suggested that CMT is best used as a diagnostic test for ovine subclinical mastitis (SCM) with a cut-off of 3 (distinct gel formation), and that automated SCC thresholds of > 1200 x 10(3) cells/ml are appropriate, especially where low prevalences are expected (e.g. < 5%). Additionally, this study showed that routine bacteriological methods were appropriate for isolation of most species of pathogen responsible for ovine SCM, but storage of samples prior to culture, either at 4 degrees C or -21 degrees C, was detrimental to the isolation of several of these organisms.

LOW‐COUNT ORGANISMS CONCEALED BY DOMINANT UROPATHOGENIC ORGANISMS IN URINE OF PATIENTS WITH ASYMPTOMATIC BACTERIURIA

We compared organisms isolated by selective culture media with those isolated by routine bacteriological methods using the same urine specimens. Urine was obtained by suprapubic bladder aspiration from 84 patients suspected of having asymptomatic bacteriuria, including 31 catheterized patients. The selective media included mannitol salt agar, Enterococcus faecalis agar, nalidixic acid cetrimide agar, and desoxycholate hydrogen sulfide lactose agar with polymyxin B or cefsulodin. Colony counts on the selective media approximated to those on heart infusion agar, except for desoxycholate hydrogen sulfide agar with polymyxin B, where the number of organisms was underestimated. In 24 patients the selective media detected a total of 32 strains not reported by the hospital laboratory. The unreported strains included coagulase-negative staphylococci (41%), other cocci (38%), enterobacteriaceae (9%) and glucose-nonfermenting gram-negative bacilli (9%) and fungi (3%), with the number of organisms being mostly < 10(5)/ml. The majority of the unreported strains coexisted with dominant gram-negative bacilli or enterococci. These results indicate that low-count organisms coexisting with dominant organisms tend to be missed by the routine bacteriological methods.

Group B meningococcal infection in children during an epidemic in Cape Town, South Africa

One hundred and thirteen children with meningococcal infection were studied during an epidemic caused by N. meningitidis group B. Fifteen per cent presented with only meningeal symptoms, the remainder showed signs of septicaemia or combined septicaemia and meningitis. Sixteen per cent of the children were in shock and 18% required admission to the Intensive Care Unit (ICU). The mortality was 4.4%. More than half the children were younger than 2 years old. There was no statistical association between the age or nutritional state of the children and any of our measures of severity. A short history of symptoms was more common in children who presented with septicaemia and severe illness, who needed admission to the ICU, or who died. Diagnosis was confirmed by routine bacteriological methods and counter-immuno-electrophoresis (CIE) in 104 children. Eighty-six per cent of the isolates were group B type 2. A history of recent antibiotic treatment was associated with fewer positive cultures, but detection of meningococcal antigen by CIE was not affected by this. CIE antigen detection was not reliable because of the high incidence of false-negative results.

Enteroinvasive Escherichia coli (EIEC) in sporadic diarrhea in Delhi.

Rectal swab/stool specimens of 104 children with diarrhea and 31 healthy controls were processed by routine bacteriological methods. Escherichia coli was grown as predominant flora on primary culture in 32 patients and 7 controls. Three representative colonies from each of these were selected at random and tested for invasive character by their ability to produce keratoconjunctivitis in guinea-pig eye and invasion of HeLa cells. Isolates from only 2 patients and none of the controls were found to be invasive. Enteroinvasive E. coli (EIEC) appear to be a relatively uncommon cause of sporadic diarrhea among children in this region.

The diagnosis of pneumococcal chest infection by counter-current-immunoelectrophoresis.

A simple, inexpensive method of counter‐current‐immunoelectrophoresis was used to detect pneumococcal antigens in sputum, serum and pleural fluid. Antigens were detected in sputa from 69% of subjects with pneumococcal pneumonia and there was a good correlation with the certainty of the clinical diagnosis. Serum antigens were found in 12.5% of subjects with either a definite or a presumed diagnosis and in one case from a patient who was not thought to have pneumococcal pneumonia. The pathogenic importance of antigens in the sputa of patients with a history of chronic respiratory disease was uncertain. Counter‐current‐immunoelectrophoresis was more sensitive than routine bacteriological methods for demonstrating the presence of pneumococci in pleural fluids.

The clinical bacteriology of purulent oral infections

The frequency of isolation of various micro-organisms from specimens of pus from oral lesions employing routine clinical bacteriological methods is described. The difficulty of establishing the significance of these organisms in oral infections is discussed and explored in regard to species of streptococci, neisseria, staphylococci, haemophili, actinomyces and bacteroides. The primacy of surgery over antimicrobial therapy in treating these infections is emphasised. Guidance on the choice of antibiotics, based on the results of antibiotic sensitivity testing is offered and the need to avoid the indiscriminate destruction of the normal oral flora stressed. The value of the nitroblue tetrazolium test is explained.