Presence Of Gram-negative Bacteria Research Articles

The first step of two-stage hip revision: what affects the result?

The aim of the study was to evaluate retrospectively the efficacy of the chronic hip PJI treatment in 2021, depending on the availability of the results of the preoperative microbiological study, and to determine the factors affecting the treatment outcomes. Material and methods: 86 patients with chronic hip PJI were assigned to groups 1 and 2, respectively, depending on the presence or absence of MBI results of preoperative biomaterials (aspirate and/or tissue biopsy) at the time of first stage. Results: The presence of the final results of the joint contents microbiological examination in the preoperative period did not affect the results of the first stage (p=0.536; OR 1.53, 95% CI 0.43-5.45). An increased risk of treatment failure was associated with presence of acetabular defects types 2C (p=0.042, OR 6.66; 95% CI 1.26-35.2) and 3B (p=0.078, OR 8.1; 95% CI 1.015-64.8), femoral defects type 3A (p=0.021, OR 6.57; 95% CI 1.49-29.01), connective tissue diseases (p=0.062, OR 5.25; 95% CI 1.05-26.2), microbial associations (p=0.02, OR 6.75; 95% CI 1.36-33.44) and Gram-negative bacteria in them (p=0.058, OR 4.2; 95% CI 1.02-17.2). As the number of risk factors increased, the probability of a negative outcome increased significantly (p0.001).Conclusion: microbial associations and the presence of Gram-negative bacteria in them, connective tissue diseases, acetabulum defects types 2C and 3B, femoral defects type 3A, as well as the cumulative number of risk factors in one patient had a significant negative impact on the outcomes of the first step of two-stage hip revision. The results of preoperative biomaterials MBI appear to be of greater importance for the PJI diagnosis than as a criterion for the choice of drugs for etiotropic antibacterial therapy. However, this assumption should be investigated in a larger sample of patients.

Bacteriospermia assessment and its relationship with conventional seminal parameters in stud dogs ejaculates (Canis familiaris)

The objective of this work was to determine the frequency of bacteriospermia and its effect on the seminal quality in canines. Dogs were divided into two groups according to weight: small dogs between 1 and 10 kg (Group 1) and dogs with more than 10 kg (Group 2). The foreskin was disinfected in each animal (n=15), and the semen sample was collected by the gloved hand method. Sperm motility, morphology, viability, and concentration were evaluated using a 40x microscope. A 10µL of semen drop was cultured by diffusion method on blood agar and MacConkey agar. Colony-forming units (CFU) were quantitatively evaluated, and biochemical identification was carried out after 48 hours at 37°C. Conventional PCR was performed on the semen samples to evaluate the presence/absence of 11 bacteria. Bacterial growth was found in all samples. The CFU/mL in blood agar were 34042.8 for group 1 and 107714.3 for group 2, while on MacConkey agar were 142.9 CFU/mL, and 21328.6 CFU/mL, respectively. Coagulase-negative Staphylococcus was the most frequent bacteria isolated by conventional culture (64.3%), and Staphylococcus aureus and Klebsiella spp. were the most common bacteria found by conventional PCR. Between both groups, only a statistically significant difference was found in normal sperm morphology. A negative correlation was observed between viability and morphology with CFU on MacConkey agar. One dog was ruled out because he had azoospermia. Canine seminal bacteriospermia is quite frequent and could alter its quality. The presence of Gram-negative bacteria is associated with greater alteration in the semen analysis.

Predictive factors of in-hospital mortality in patients with laboratory-confirmed Escherichia coli, Klebsiella species or Pseudomonas aeruginosa bloodstream infections.

Gram-negative bloodstream infections (GNBSI) are confirmed by the presence of gram-negative bacteria in the bloodstream and pose a significant healthcare issue as they increase the risk of sepsis and mortality. In England, the aim is to reduce GNBSI cases and further deterioration through enhanced population surveillance of patients with a laboratory-confirmed GNBSI to inform on healthcare policies. The objective of this study was to evaluate the factors associated with in-hospital mortality in patients with a laboratory-confirmed Escherichia coli, Klebsiella or Pseudomonas aeruginosa GNBSIs, with data obtained from the enhanced data capture for the surveillance of GNBSIs. All patients with a laboratory-confirmed GNBSI at a single centre, admitted between April 2017 and March 2019, were included in this retrospective observational study. Demographic and recent exposure to healthcare risk factors were collected and assessed for the association with in-hospital mortality. In 1113 patients with laboratory-confirmed GNBSIs, the in-hospital mortality rate was 13%. Multivariable analysis confirmed that patients with respiratory (OR = 3.73, 95%CI = 2.05–6.76), gastrointestinal (2.61; 1.22–5.58) or skin (3.61; 1.24–10.54) infection primary focus had a greater risk of in-hospital mortality, compared to upper urinary tract infections. Increased risk of in-hospital mortality was also observed in patients with hospital-onset GNBSIs (OR = 1.87; 1.17–2.97) compared with community-onset healthcare acquired GNBSIs, or who were on dialysis at the time of the GNBSI (3.28; 1.01–10.14), as well as in patients who had recently been discharged from hospital (1.55; 1.01–2.38), or had a vascular device recently manipulated (2.41; 1.01–5.74). Results confirm that the data obtained from the enhanced data capture for GNBSIs in England can predict in-hospital mortality in patients with a GNBSI. Several factors associated with an increased risk of in-hospital mortality have been identified. Results should be reported back to clinicians in order to identify patients at a greater risk of dying in-hospital who may benefit from further monitoring.

Rapid Detection of Pathogenic Bacteria by the Naked Eye.

Escherichia coli O157:H7 and Staphylococcus aureus are common pathogens. Gram-negative bacteria, such as E. coli, contain high concentrations of endogenous peroxidases, whereas Gram-positive bacteria, such as S. aureus, possess abundant endogenous catalases. Colorless 3,5,3′,5′-tetramethyl benzidine (TMB) changes to blue oxidized TMB in the presence of E. coli and a low concentration of H2O2 (e.g., ~11 mM) at pH of 3. Moreover, visible air bubbles containing oxygen are generated after S. aureus reacts with H2O2 at a high concentration (e.g., 180 mM) at pH of 3. A novel method for rapidly detecting the presence of bacteria on the surfaces of samples, on the basis of these two endogenous enzymatic reactions, was explored. Briefly, a cotton swab was used for collecting bacteria from the surfaces of samples, such as tomatoes and door handles, then two-step endogenous enzymatic reactions were carried out. In the first step, a cotton swab containing bacteria was immersed in a reagent comprising H2O2 (11.2 mM) and TMB for 25 min. In the second step, the swab was dipped further in H2O2 (180 mM) at pH 3 for 5 min. Results showed that the presence of Gram-negative bacteria, such as E. coli with a cell number of ≥ ~105, and Gram-positive bacteria, such as S. aureus with a cell number of ≥ ~106, can be visually confirmed according to the appearance of the blue color in the swab and the formation of air bubbles in the reagent solution, respectively, within ~30 min. To improve visual sensitivity, we dipped the swab carrying the bacteria in a vial containing a growth broth, incubated it for ~4 h, and carried out the two-stage reaction steps. Results showed that bluish swabs resulting from the presence of E. coli O157: H7 with initial cell numbers of ≥ ~34 were obtained, whereas air bubbles were visible in the samples containing S. aureus with initial cell numbers of ≥ ~8.5 × 103.

Isolation and Identification of Uropathogenic E. coli and its Antimicrobial Susceptibility Pattern with Special Reference to Extended Spectrum Beta-Lactamases (Esbl)

Urinary tract infections (UTI) are the most common bacterial infection among the humans. One of the most important factors impacting the management of UTI over the past decade is emergence of anti-microbial resistance among uropathogens. ESBL production is one of the most common mechanisms of anti-microbial resistance, the other being Amp C β-lactamases in gram negative bacteria. This study was undertaken to establish the prevalence of ESBL producing strains and their anti-microbial susceptibility pattern to newer agents to guide therapy for urinary tract infection. The present studies isolates and identify Uropathogenic Escherichia coli and its antibiotic susceptibility pattern with special reference to ESBL. The emergence of β-lactamase is a Metter of serious concern .The drug resistance in gram negative bacilli is due to production of β-lactamases, AmpC lactamases, Efflux mechanisms and Porin deficiency. Out of the total 3580 urine samples, 987 samples (27.56%) showed No growth, 1786 (49.88%) showed the presence of Gram negative bacteria. Totally, 1081 E. coli species were isolated and < 30% of them were found to be ESBL positive. Uropathogenic E. coli isolates are highly susceptible towards Meropenem and Imipenem antibiotics and highly resistant towards β-lactam and Cephalosporins antibiotics. Hence, the present study urged to implement the management plan for using those antibiotics in patients for preventing the antibiotic resistance.

Prognostic value of intratumoral Fusobacterium nucleatum and association with immune-related gene expression in oral squamous cell carcinoma patients

Changes in the oral microbiome, particularly Fusobacterium nucleatum, are associated with oral squamous cell carcinoma (OSCC). F. nucleatum has been reported to modulate local immunity in cancers. We aimed to assess the association between intratumoral F. nucleatum and clinico-pathological features, relapse, and overall survival (OS) in two independent cohorts of patients with OSCC, and to explore the interplay with immune-related genes. We retrospectively analyzed tissue samples from a first cohort of 122 patients with head and neck squamous cell carcinoma, including 61 OSCC (cohort #1), and a second cohort of 90 additional OSCC (cohort #2). We then performed a sensitivity analysis on the merged cohort of OSCC patients (N = 151). F. nucleatum 16S rRNA gene sequences were quantified using real-time quantitative PCR. The presence of gram-negative bacteria and macrophages was confirmed by LPS and CD163 immunostainings, respectively. F. nucleatum positivity was associated with older age, less alcohol and combined alcohol plus tobacco consumption, and less frequent lymph node invasion. There was a trend for a lower recurrence rate in F. nucleatum-positive cases, with less metastatic relapses compared to F. nucleatum-negative tumors, and significantly longer OS, relapse-free and metastasis-free survival. F. nucleatum status was independently associated with OS in multivariate analysis. Immune-related gene and immunohistochemistry analyses showed that gram-negative bacteria load inversely correlated with M2 macrophages. F. nucleatum-associated OSCC has a specific immune microenvironment, is more frequent in older, non-drinking patients, and associated with a favorable prognosis.

Source and antibiotic susceptibility of gram-negative bacteria causing superficial incisional surgical site infections

BackgroundSurgical site infections (SSIs) are a major public health problem worldwide and the second-most frequently reported type of nosocomial infection. The presence of Gram-negative bacteria (GNB) in the hospital environment is significantly associated with SSIs. As such, this study aimed to explore the rate, antibiotic profile and common sources of bacteria in SSI. MethodsA cross-sectional study was conducted between August 2018 and July 2019 in three surgical centers in Kalar city, Kurdistan region, Iraq. A total of 512 patients, who underwent surgery were followed up with for superficial incisional SSI. Pre-, intra- and postoperative swabs were collected from patients and the surrounding hospital environment and processed for isolation and identification of GNB by microbiological and molecular methods. The isolates were typed by sequencing and tested for antibiotic resistance using the disc-diffusion technique. ResultsSSI developed in 113 (22.07%) of the patients studied. GNB were involved in 48 (9.38%) cases; among these, Klebsiella spp. were the predominant cause 21/48 (43.75%), followed by Escherichia coli 14/48 (29.17%), Pseudomonas aeruginosa 7/48 (14.58%), Enterobacter spp. 3/48 (6.25%) and A. baumannii 2/48 (4.17%). Citrobacter koseri showed the lowest rate of infection 1/48 (2.08%). Sequencing analysis and the antibacterial resistance profile revealed that 25 (74.28%) of the 48 SSI isolates were from the hospital environment, whereas the rest were exogenous, with an undetermined source. The involved GNB were highly resistant to most antibiotics tested. ConclusionSSIs caused by GNB were mostly exogenous or from the hospital environment, not from endogenous sources. All of the bacterial isolates detected from SSI patients were multidrug resistant.

The Effect of Packaging Materials Type and Addition of Turmeric (Curcuma Domestica) Flour on Calf Starter Pellet to The Total of Fungi, The Existence of Gram Negative Bacteria and Aspergillus Flavus

The purpose of the research was to examined the effect of packaging material and the addition of turmeric levels on the calf starter pellet to the total fungi, the presence gram negatives and Aspergillus flavus at 56 days. The method was used the pelleting process. The research used a CRD with factorial pattern (3x2) and 3 replications. The first factor was the addition of turmeric flour (w/w) (0%, 0.6% and 1.2%) and the second factor was the type of packaging material (plastic and paper). Data on the total of fungi were analyzed using ANOVA, while data on the presence of gram negative bacteria and Aspergillus flavus were analyzed descriptively. The results showed that there was no interaction effect between the first factor and the second factor to the total fungi. Gram negative bacteria were still found on the pellets in all treatments. Whereas Aspergillus flavus was only found at the level 0%(plastic) and 0,6%(paper). The conclusion was the addition of turmeric levels in the calf starter pellet who were packaged with plastic and paper have not been able to inhibit the growth fungi and gram negative bacteria, but can inhibit Aspergillus flavus at the turmeric flour level of 1,2%.

Short communication: Coliform Petrifilm as an alternative method for detecting total gram-negative bacteria in fluid milk

Postpasteurization contamination (PPC) of fluid milk remains a challenge for some dairy processors. Pseudomonas is the most common contaminant of fluid milk after pasteurization, and therefore methods to detect PPC should be inclusive of Pseudomonas and other gram-negative contaminants (e.g., coliform bacteria). Our objective was to compare the ability of 3M (St. Paul, MN) coliform and Enterobacteriaceae (EB) Petrifilm to detect total gram-negative bacteria with that of the standard method, crystal violet tetrazolium agar. To that end, we evaluated coliform Petrifilm, EB Petrifilm, and crystal violet tetrazolium agar to detect gram-negative bacteria in naturally contaminated samples of fluid milk. A total of 92 observations derived from shelf-life testing of 33 milk samples from 5 different processing facilities were evaluated for (1) presence of coliforms on coliform Petrifilm at both 24 and 48 h of incubation; (2) presence of any growth, regardless of gas production, on coliform Petrifilm at both 24 and 48 h of incubation; (3) presence of EB on EB Petrifilm at both 24 and 48 h of incubation; (4) presence of any growth, regardless of gas or acid production, on EB Petrifilm at both 24 and 48 h of incubation; and (5) presence of gram-negative bacteria on crystal violet tetrazolium agar after 48 h of incubation. Sensitivity and specificity analysis of results indicated that compared with the standard method (i.e., crystal violet tetrazolium agar), the method that performed the best, based on balanced accuracy (i.e., the average of sensitivity and specificity), was coliform Petrifilm evaluated for the presence of any growth after 48 h of incubation (sensitivity = 0.787; specificity = 0.839). This method can be easily adopted by the dairy industry as many processing facilities already test for coliforms using coliform Petrifilm. Improving the ability of processors to detect PPC will improve the quality of the fluid milk supply.

Detection of Pseudomonas aeruginosa in Dead Chicken Embryo with Reference to Pathological Changes and Virulence Genes

This work was designed to investigate incidence of P. aeruginosa in dead in shell chicken embryos and molecular detection of virulence genes and study their pathogenicity effect on embryonated chicken eggs and chicks. Out of 100 pooled samples were collected from liver, heart and yolk sac of dead in shell chicken embryos, a total of 24 (24%) P. aeruginosa isolates were isolated.On carrying PCR test for 24 P. aeruginosa isolates there were high distribution of (opr L) and ( lasI ) genes with 100% while distribution of other virulence genes was as follows: toxA (83.3%), lasR (75%), Rhl1 (70.8%). Pathogenicity of P. aeruginosa on embryonated chicken eggs was studied either by inoculation of P. aeruginosa strain via yolk sac route in a dose of 0.1 ml suspension 14x10⁷. Which gave 100% embryonic mortalities after 3 days or by dipping group of 17 days –old embryonated chicken eggs in a dose of 6.1X 109 /ml which gave 90% embryonic mortality. Experimentaly infected chicks showed 70% mortality. Histopathological changes in dead embryos from infected embryonated eggs and sacrificed, dead infected chicks revealed lymphocytic hepatitis with presence of gram negative bacteria. Lymphocytic nephritis with hemosiderosis found in renal parenchyma. Heart showed lymphocytic myocarditis. Lung showed alveolar emphysema, interstitial pneumonia and perivascular fibrosis. P. aeruginosa was re-isolated from internal organs of sacrificed hatched chicks and from liver and yolk sac of dead embryos.

Identification of gram-negative bacteria of Bali cattle with repeat breeding cases on East Lombok, West Nusa Tenggara Province

The target of self-sufficiency in 2017 has been launched by President of Republic Indonesian. The province of West Nusa Tenggara (NTB) with “Bumi Sejuta Sapi (BSS) and UPSUS SIWAB Program has been recognized as the key site for production and supply of meat and cattle for all of part of Indonesia. One of the classic problems that led to the lack of that program is the repeat breeder in cattle that associated with the presence of Gram-negative bacteria in the reproductive tract. The purpose of this study was to identify bacteria in the reproductive tract of Bali cattle that used for UPSUS SIWAB Program in East Lombok. This research was conducted in January 2019 at Kopang Village, East Lombok district. Four Bali cattle with repeat breeders case in east Lombok were used in this research. Uterine fluid as samples were collected with a sterilized plastic sheet of artificial gun. The sterilized plastic sheet of artificial gun was inserted into the vagina and then passed through the cervix up to the body/horn of the uterus. The end of the plastic sheet was cut and placed into BHI (Brain Heart Infusion) and they were incubated over the night. After 24 hours the samples were inoculated in MacConkey agar under aerobic conditions at Laboratory of Public Health and calibration, Province of West Nusa Tenggara Barat. The colonies were purified and characterized by gram staining and biochemical methods. The result of this study showed that three pathogens bacterial, including Proteus vulgaris, Escherichia coli, and Citrobacter freundii were identified from Uterine fluid of four Bali cattle.

Frequency and antibiotic sensitivity of gram negative bacteria isolated from raw fish sold in Karachi, Pakistan

Fish serve as an important source of nutrition and also acts as a reservoir for different pathogenic organisms. Sewage pollution in the water bodies is the major source of pathogenic bacteria in seafood. In this study, the presence of Gram negative bacteria in raw fish samples was detected and the antibiotic susceptibility pattern of the isolates was determined.A total of 25 raw fish samples were collected at random from different local markets of Karachi in sterile zip-lock plastic bags. From these samples, a total of 73 Gram negative bacterial strains were isolated. Of these 73 Gram negative isolates, 6 were E. coli, 14 K. pneumoniae, 7 E. aerogenes, 4 E. cloacae, 5 C. freundii, 1 C. koseri, 2 P. vulgaris, 3 P. mirabilis, 7 Salmonella spp., 6 Shigella spp., 2 Serratia spp., 2 Pseudomonas spp., 7 Yersinia spp., 3 Aeromonas spp. and 4 were Vibrio spp. The most abundant species among all the isolated Gram negative bacteria was Klebsiellapneumoniae(19.17%) followed by Enterobacter aerogenes(9.59%), Salmonella spp. (9.59%) and Yersinia spp. (9.59%). Antibiotic susceptibility pattern of all the Gram negative isolates was determined by Kirby-Bauer disc diffusion method in which a panel of eight antibiotics was tested. The incidence of resistance was highest against Cefazolin (93.2%) and lowest resistance was observed against Gentamicin (1.4%). Of the 73 Gram negative isolates, 24 were found to be multi-drug resistant (MDR). In conclusion, the bacteriological analysis of raw fish samples showed that the quality of raw fish was poor as high bacterial load was obtained and enteric bacteria were isolated from most of the samples indicating fecal pollution. This study provides valuable information about the presence of MDR Gram negative pathogens in raw fish sold locally which could be useful in the selection of antibiotics for empirical treatment. Keywords: Enteric bacteria; Fresh fish; Karachi; Multidrug resistant; Pre-enrichment http://dx.doi.org/10.19045/bspab.2019.80105

Microbial contamination of coffee during postharvest on farm processing: A concern for the respiratory health of production workers

Coffee workers respiratory health problems, found to be associated with dust exposure in the coffee factories, but the content of the dust is not well known. A cross-sectional survey was conducted to assess the microbial contamination of coffee in dry and wet coffee process, from four farms in two regions of Ethiopia. A total of 36 samples of coffee were collected for laboratory investigation. The microbial load in the dry process ranged from 6.9 × 102 to 7.2 × 105 colony forming units (CFU)/mL while the microbial load in wet process ranged from 2.5 × 102 to 4.6 × 105 CFU/mL. The results indicate the presence of gram negative bacteria in dried and stored beans from both the wet and dry process. During further coffee processing possible release of endotoxin from coffee contaminated by gram negative bacteria might affect coffee workers respiratory health. Further studies are required to assess the relation between bacterial contamination of coffee and endotoxin level in coffee factories.

The sorption of organic compounds from water during technological start-up of carbon filters

Natural waters contain organic substances that have a major impact on the quality of water, in particular on its organoleptic properties. They can also change its physical and chemical properties and have a negative impact on the technological processes used during water treatment. There is no doubt that organic substances contained in natural waters have to be removed during treatment processes in order to achieve concentrations consistent with the requirements concerning the quality of water intended for human consumption. A very effective method of organic compounds elimination from water is to use of biologically active filters (BAF). This article presents the results of pilot scale research on the effectiveness of removal of organic compounds from water using filtration through a bed of activated carbon filters, during their technological start-up. The obtained results showed high, more than 91% efficiency of reducing the content of organic compounds expressed by TOC, COD (KMnO4) and UV254 absorbance. The predominant process at the start of the use of carbon filters has been sorption. During the research, the development of biofilm in the filter bed was also controlled. The identification of microorganisms showed the presence of Gram-negative bacteria in the beds of both filter columns.

Does chlorhexidine mouthwash reduce the rate of oral colonization bygram-negative bacteria in patients with chemotherapy? Aplacebo-controlled trial

The presence of gram-negative bacteria in the oral cavity is an undesirable occurrence in patients undergoing chemotherapy. Our aim was to investigate the antibacterial effect of 0.12% chlorhexidine mouthwash in chemotherapypatients with a randomized, double-blind, placebo-controlled trial. There were no significant differences between oral colonization rates; there may be local factors that interfere with chlorhexidine activity.

P15.09: Association of abnormal vaginal colonisation and preterm delivery: effect of cervical length

The exact relationship between abnormal vaginal colonisation and adverse pregnancy outcome in preterm labour remains unclear. Therefore, we hypothesised that women with shorter cervical length (CL) are predisposed to ascending infection in women with preterm labour (PTL) or short CL. We aimed to examine abnormal vaginal colonisation rate according to cervical shortening and to compare the association between abnormal vaginal colonisation and spontaneous preterm delivery (SPTD) before 34 weeks according to CL in women with PTL or short CL. This study included 580 patients who admitted to our high-risk unit due to PTL or short CL and underwent vaginal culture and measurement of CL at admission between 2005 and 2015. Maternal age, parity, parity, body mass index and CL at admission, vaginal culture result, birth weight of neonates, and SPTD before 34 weeks were retrospectively reviewed. The study population was divided three groups according to the degree of cervical shortening; CL < 0.5cm (n=113), 0.5cm to 1.5cm (n=145), ≥ 1.5cm (n=322). There was no difference in abnormal vaginal colonisation rate according to the CL shortening (17.7%, 17.9%, 13.7% in group with CL < 0.5cm, 0.5cm to 1.5cm, ≥ 1.5cm, respectively, p=0.22). Overall, the rate of SPTD was not different according to the presence or absence of abnormal vaginal colonisation in three groups (93.3% vs 68.3%, p=0.06 in group with CL < 0.5cm; 40.9% vs 55.7% p=0.21 in group with 0.5cm to 1.5cm, 42.4% vs 33.2% p=0.30 in group with CL ≥ 1.5cm). However, in group with CL < 0.5cm, the presence of gram negative bacteria (but not gram positive) was significantly associated with higher SPTD (100% vs 68.6%, p=0.03). In other two groups, there was no difference in the rate of SPTD according to either the presence of gram positive or negative bacteria. Our data showed that maternal vaginal colonisation by gram-negative bacteria in advanced cervical shortening in PTL or short CL is associated with SPTD before 34 weeks. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract 5136: Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues

Abstract There is currently a great interest in understanding direct interactions between human-associated microbial populations and the tumor microenvironment. In this study, we aimed to develop a chromogenic in situ hybridization (CISH) assay for universal detection of bacteria in formalin fixed paraffin embedded (FFPE) cancer tissues. This was accomplished using the RNAscope assay (Advanced Cell Diagnostics) with a CISH probe set designed to target the most highly conserved region in the bacterial 16S ribosomal RNA (rRNA) gene. Optimization of the 16S rRNA CISH assay was conducted using FFPE mouse colon, Mycobacterium tuberculosis infected rabbit lung, and Propionibacterium acnes infected mouse prostate tissues. Positive control (peptidylprolyl isomerase B- PPIB) and negative control (maize –Zm) stains were also conducted on the tissues. We additionally developed immunohistochemistry (IHC) assays with antibodies against lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to visualize gram negative and gram positive bacteria, respectively, as a means to verify the results of the CISH assay. As our laboratory has a specific interest in the interactions between prostate infections and prostate cancer, we used this technique to interrogate a series of 10 radical prostatectomy specimens that contained a very high degree of acute and chronic inflammation, and were suspicious for the presence of an infectious organism. Both CISH and IHC were conducted in FFPE radical prostatectomy samples. The 16S rRNA CISH assay detected bacteria in all of the control tissues. Interestingly, 3 of the 10 human radical prostatectomy samples that we examined showed bacterial 16S rRNA signal concentrated in areas with acute inflammation where the glandular lumens were filled with neutrophils. Two of the radical prostatectomy samples that showed positive bacterial 16S rRNA CISH signal also showed positive LPS staining indicative of the presence of gram negative bacteria. The remaining positive sample was suspicious for a gram positive intracellular organism. This study shows preliminary data supporting robust detection of bacteria in a subset of human prostate samples. We expect the frequency of bacterial infections of this nature present in radical prostatectomy specimens would be low, and that we were only able to identify these cases because we screened for samples with very high levels of inflammation. Detection of both bacterial RNA and protein in the tissues corroborates the strength and reliability of the 16S rRNA CISH. The use of these two methods concurrently can provide valuable information on bacterial presence in a range of clinical samples. In the future, we plan to use these methods to probe for bacterial presence and in understanding the spatial dynamics of the urinary microbiome. We expect that this method may also be of broader use in the study of many infection-associated cancers. Citation Format: Eva Shrestha, Karen S. Sfanos. Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5136.

Occurrence of Gram-negative bacteria in cockroaches obtained from different areas of Dhaka city

The presence of Gram-negative bacteria in cuticle and alimentary tract of cockroaches collected from four selected areas of Dhaka city was investigated. Using detection key, a total of 12 cockroaches were identified as Periplaneta americana (58.33%), Blattella germanica (33.33%) and Blatta orientalis (8.33%). From these three species 54 different bacterial isolates were obtained. Out of them, 33 were obtained from P. americana (14 from external and 19 from gut), 9 from B. orientalis (5 from external and 4 from gut) and 12 from B. germanica (5 from external and 7 from gut). Bacterial isolates obtained from external surface of the cockroaches belonged to four different Gram negative bacterial species (Klebsiella spp., Pseudomonas spp., Enterobacter spp. and E. coli), six bacteria have been obtained from alimentary tract (Klebsiella spp, Pseudomonas spp., Enterobacter spp., Escherichia coli, Salmonella spp. and Shigella spp.). Pseudomonas spp. were the most predominant bacteria isolated from external surface (33.34%) while Klebsiella spp. were found to dominate in alimentary tract (23.33%). P. americana carried the highest number (33) of bacterial isolates and B. orientalis carried the minimum (9). This study shows that cockroaches carry several bacteria on their external body surface and alimentary tract and can play a role in bacterial transmission to humans. Therefore, the existence of cockroaches in human dwellings is a serious public health problem.Bangladesh J. Zool. 44(2): 229-236, 2016

Antimicrobial Resistance of ESBL Producing Coliforms Isolated from Retail Meat Samples

Extended Spectrum Beta lactamases (ESBLs) are special enzymes which are found in groups and can hydrolyze the third generation cephalosporins. Their Production is the commonest cause of resistance to beta-lactam antibacterial agents among Gram-negative bacteria. There is spread of drug resistance among food animals due to the use of antimicrobials. Pathogens develop resistance in their animal reservoirs and then these resistant strains are transmitted to humans where they may cause infections which are difficult to treat. In this study the antimicrobial resistance due to ESBL producing coliforms in humans and in edible animal meat was examined. The main objective of this study was to determine the fact that a substantial increase in antimicrobial resistance posed by ESBL producing coliforms in humans is due to edible animal meat. Various chicken, meat and beef samples were collected from different sites in Faisalabad. The samples were enriched in Trypticase Soy Broth and were streaked on Nutrient Agar then on MacConkey Agar to check the presence of Gram negative bacteria. Gram Negative isolates were isolated by gram staining and biochemical test (Triple Sugar Iron; TSI). Antibiotic susceptibility testing was performed through disc diffusion method. In which 28% isolates were resistant to Cefixime, 20% to Ceftriaxone, 24% to Clavulanic acid and 30% to Cefotaxime. As the next step, a set of PCR was optimized for amplification of major genes of ESBL conferring resistance to selected antibiotics. As a result 20% Bla (CTX-M), 4% Bla(OXA), 2% Bla(PER) and 1% Bla(Ges) were amplified through PCR. These are the major genes of ESBL which are going to become more and more resistant towards the first, second and third generation cephalosporins.

Procalcitonin Is a Marker of Gram-Negative Bacteremia in Patients With Sepsis

BackgroundPrediction of the species of pathogen among patients with sepsis within hours would be helpful in accelerating proper treatment. As a potential method of shortening the time to identification, this study considered the usefulness of measuring procalcitonin (PCT) to predict blood culture (BC) results. MethodsThe authors retrospectively analyzed the data of patients with a diagnosis of sepsis in their hospital from December 2012 to December 2013. The authors analyzed all diagnostic episodes consisting of BC and PCT concentration. The diagnostic performance of PCT to predict gram-negative bacteremia was tested using a receiver operative characteristic curve. Logistic regression was constructed using the presence of gram-negative bacteria as the dependent variable. ResultsA total of 262 diagnostic episodes met the inclusion criteria. According to BC classifications, a significantly higher value of PCT was observed in bloodstream infections caused by gram-negative bacteria (26.7 ng/mL, 0.09–188.3) than that in bloodstream infections caused by gram-positive bacteria (0.84 ng/mL, 0.05–18.79) or Candida spp. (1.12 ng/mL, 0.07–49.68). A cutoff value of ≥3.39 ng/mL for PCT showed a sensitivity of 80%, a specificity of 71%, a positive predictive value of 35%, a negative predictive value of 91% and an area under the curve of 0.73 for gram-negative bacteremia identification by BC. Among the 122 diagnostic episodes with positive BC results, a cutoff value of ≥6.47 ng/mL for PCT yielded a sensitivity of 74%, a specificity of 81%, a positive predictive value of 82%, a negative predictive value of 75% and an area under the curve of 0.81 for gram-negative bacteremia identification. ConclusionsPCT may represent a useful tool for differentiating gram-positive from gram-negative bloodstream infection with a significantly higher PCT level indicating gram-negative bacteremia.