Agar Base Research Articles

Use of the Micro-Agar Larval Development Test to Differentiate Resistant and Susceptible Cooperia spp. Isolates in Cattle Within the Context of Parasite Population Replacement.

Gastrointestinal nematode infections are a global concern in grazing cattle production systems, even more so due to the widespread problem of anthelmintic resistance. In response, early anthelmintic resistance detection methods, such as the micro-agar larval development test (MALDT), and parasite management strategies, such as the replacement of resistant parasite populations with susceptible ones, have been developed. This study aimed to characterize ivermectin-susceptible and -resistant isolates of Cooperia spp. using MALDT in the context of a parasite population replacement strategy. Three Cooperia spp. field isolates were evaluated: a susceptible one (Coop-S), a resistant one (Coop-R), and a post-replacement one (Coop-PR). The MALDT was performed in 96-well plates with 12 known concentrations of eprinomectin (EPR) on an agar base. Each test was performed in quadruplicate. Data analysis included nonlinear regression to determine EC50, EC90, and EC99 values, resistance ratios (RRs), and R2. The results showed clear differentiation between the isolates, with RR values of 5.78 and 1.28 for Coop-R and Coop-PR, respectively, compared to Coop-S. The MALDT proved to be a reliable tool for differentiating ivermectin-susceptible from ivermectin-resistant isolates of Cooperia spp., and future evaluations of this test in mixed nematode populations are recommended for routine diagnosis of anthelmintic resistance.

Pathological and Fungal Isolation study of dog skin disease in Iraq Governorates

Dogs are susceptible to several types of infectious diseases can be worrisome for their owner because some of them are fatal if not treated soon enough. Fungal infection can be contracted through different parts of body furthermore many of them are zoonotic infection among human and dogs the signs of any disease typical depend on location of infection either local or system infection. Local infection usually involves skin surface that look like wound the objective of this study was to briefly about the most common pathogenic fungal infection in dogs by describing information about the causative agent, clinical signs and grossly appearance with histopathological change skin examination current study were done on 200 dogs collected in Iraq governorates (Baghdad, Erbil, Anbar, Sulaymaniyah) during a period 6 months (January – June) 2023. 200 examined dogs with different age, breed and sex ( k9, ornamental and stray dogs ) were observed fungal isolation and pathological examination with clinical signs by using diagnostic tools for skin biopsies (biopsy punch) from suspected infected skin dogs. Results showed pathogenic fungi after collected on fungal culture for detection pathogenic fungal (Dermatophyte Test Agar) D.T.M Agar Base. Fungal isolation constitutes the following pathogenic fungi (100 infected samples from 200 suspected samples). 60% Aspergillus niger, 30% Trichophyton Verrucosum, 10% Alternaria spp. Clinical signs and grossly lesion showed complete skin ulcer, allopecia, odema, abscessation while microscopic appearance revealed skin ulcer, hyperkeratosis, folliculitis, necrosis, apoptosis, pyogranuloma and granulation tissue with septate hyphae in skin dermis and epidermis which infected with Aspergillus niger.

Cd2+ Sequestration by Ureolytic Bacteria Isolated from Goat Faeces and Nursery Wastewater

As modern industrial production makes extensive use of heavy metals, a significant amount of sewage and solid wastes containing heavy metals are released into the environment. There is significant potential for bioremediation of multiple heavy metal contamination using biomineralization to immobilise the toxic metal. In this study, microbially induced carbonate precipitation (MICP) technology was used to treat Cd2+ contamination on plants, humans, and the environment. Ureolytic bacteria that collected from goat faeces and nursery wastewater were stimulated using enrichment technique. Next, physicochemical properties of the bacteria including urea agar base test, biomineralization test, conductivity & pH measurement test and optical density test were examined through enrichment subculturing of the bacteria. The morphological characterization of precipitate formed from both samples were analayzed by using scanning electron microscopy- energy dispersive x-ray diffraction method. Ureolytic bacteria of the goat faeces sample demonstrated greater tolerance to Cd2+ concentration compared to the nursery wastewater sample by obtaining a higher optical density values and larger amounts of precipitate produced by the goat faeces sample across all tested Cd2+ concentrations. As a result, from the atomic absorption spectrophotometry (AAS) analysis, the goat faeces sample showed higher efficiency in removing Cd2+ compared to the nursery wastewater sample, with a removal efficiency of 89.06% for the goat faeces sample and 77.75% for the nursery wastewater sample. Hence, this study confirmed that the interaction between ureolytic bacteria isolated from waste sources and heavy metal ions is vital for the overall effectiveness of heavy metal removal.

Estimation of IL-8 and TNF-α Levels in Pediatric Diarrhea Patients Infected with Enterohemorrhagic E. coli O157:H7

Abstract Background: Verotoxins are bacterial virulence factors produced by E. coli O157:H7, transmitted by the fecal-oral route. Objectives: The aim of this article was to diagnose E. coli O157:H7 which causes diarrhea and sometimes develops into HUS, which considers pig health problems and estimates the levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α in the sera of pediatric patients infected with Enterohemorrhagic E. coli compared to the control group. Materials and Methods: Stool and blood samples were collected from 421 pediatric patients with diarrhea, ranging in age from birth to 13 years old, from March to October 2022. Samples were collected from Al Noor Teaching Hospital, Babylon Hospital for Pediatric and Gynecology, Hilla, Iraq. E. coli O157:H7 was cultured on eosin methylene blue (EMB) and Sorbitol MacConkey agar (SMA), confirmed by biochemical test and cultured on HiCrome E. coli O157:H7 selective medium which was an agar base supplemented with cefixime tellurite agar. Serum from 30 pediatric diarrhea patients infected with E. coli O157:H7 compared with 30 healthy children as control group used to determine serum levels of IL-8 and TNF-α by sandwich ELISA. Results: The results revealed that out of the total 421 samples used in this study, E. coli O157H:7, represented 7% (30 of 421) stool samples. This 30serum samples of infected children as well as 30 samples from healthy children subjected to the estimate serum level of IL-8 and TNF-α which record significant differences P ≤ 0.01 and P ≤ 0.05 to this cytokines in different age group; the mean of IL-8 level was 283.62 ± 17.8 pg/mL (7–9 years), and the mean of TNF-α was 208.62 ± 28.7 pg/mL (10–13 years) comparative with the control group of 80.58 ± 15.4pg/mL and 32.50 ± 7.5 pg/mL, respectively, and also result showed an increased mean level of IL-8 than TNF-α in the male comparative with female 195.19 ± 10.4 pg/mL and 159.05 ± 12.4 pg/mL, respectively, comparative with the control group. The result showed no significant differences in IL-8 and TNF-α between watery diarrhea (192.43 ± 24.3 pg/mL and 136.05 ± 20.4 pg/mL) and bloody diarrhea (189.02 ± 22.5 pg/mL and 123.80 ± 13.5 pg/mL), and also result showed significant increase of mean sera level of IL-8 than TNF-α in formula feeding children comparative with breastfeeding children (187.87 ± 19.5 pg/mL and 119.93 ± 17.4 pg/mL, respectively). Conclusion: The finding of this study suggested that increased levels of IL-8 and TNF-α are present in all age groups, in male comparative with female, and also in pediatric diarrhea feeding by formula than breastfeeding and no differences of this cytokine according to consistency of diarrhea. These results contribute to using the immune profile as a serological marker for diagnosing diarrhea caused by E. coli O157:H7 in comparison with the control group.

ISOLATION OF BACTERIA CAUSING DENTAL CARIES FROM THE CLINICS OF THE COLLEGE OF BILAD AL-RAFIDAYN UNIVERSITY

This study deals with Isolation of bacteria causing dental caries from the Forty samples (male and female) were collected from patients attending the clinics of the Department of Dentistry at Bilad Al-Rafidayn University College for the period 7/11/2022 to 4/5/2023. The total of (58) isolates before and after cleaning and tooth extraction collected from male and female samples .After diagnosing these isolates, it was found that. Streptococcus (42.5%) and gram-negative (20%) before dental work and cleaning . The Percentage of Streptococcus after cleaning (30%) and the gram-negative rod (7.5%). The percentage of Streptococcus isolated from tooth extraction (20%) and staphylococcus (2.5%) and gram-negative rod(2.5%). The spacemen were collected from the oral cavity and the culture was on mitis salivarius(Himedia/India) agar base, that was determined by biochemical tests by use of blood agar to detect hemolysis, the results revealed the samples were positive.

Performance evaluation of loop-mediated isothermal amplification, polymerase chain reaction and real-time polymerase chain reaction methods to detect Neisseria gonorrhoeae among symptomatic patients from India.

Background: Neisseria gonorrhoeae is one of the most important causative organisms in causing sexually transmitted infections. The clinical presentation of gonorrhoea mimics the symptoms of other sexually transmitted infections, and a proper diagnosis of the same is therefore crucial in patient management. The current study intended to compare different in-house molecular methods: that is, conventional PCR, real-time PCR, and LAMP assay for detection of N. gonorrhoeae. Methods: A total of 163 samples were collected from 145 patients who presented with urethral and vaginal discharge. Collected samples were processed for culture on GC agar base, and three different molecular diagnostic tests (conventional PCR, real-time PCR, and LAMP assay) were performed simultaneously on all the samples. Results: Culture of N. gonorrhoeae was positive in 17 out of 21 (80.9%) swab samples. With culture as the gold standard method, conventional and real-time PCR had a sensitivity of 94.1%, whereas the sensitivity of the LAMP assay was found to be 88.2%. All three methods had a specificity of 100%. In addition to swab samples, evaluation of urine samples by different molecular methods yielded a good concordance with a kappa value of 0.85 by conventional PCR and real-time PCR showing a perfect level of agreement, while the LAMP assay was found to have a substantial level of agreement. Conclusion: LAMP assay had a comparable diagnostic accuracy to other molecular methods for the detection of N. gonorrhoeae and can be used as a point-of-care test in resource-limited settings.

Detection of Drug Resistance Staphylococcus Aureus from Sheep Mastitis Milk

Detection of Drug Resistance Staphylococcus Aureus from Sheep Mastitis Milk

An emerging zoonosis: molecular detection of multidrug-methicillin resistant Staphylococcus aureus from butchers' knives, livestock products and contact surfaces.

Methicillin-resistant Staphylococcus aureus (MRSA) transmission in livestock, community, and healthcare settings poses a significant public health concern both locally and globally. This study aimed to investigate the occurrence, molecular detection, and antibiogram of the MRSA strain in fresh beef, contact surfaces, and butchers' knives from the four major abattoirs (Karu, Gwagwalada, Deidei, and Kubwa) located in the Federal Capital Territory, Nigeria. A multi-stage sampling technique was used to collect 400 swab samples from butchers' knives (132), fresh beef (136), and contact surfaces (132). Presumptive colonies on mannitol salt agar were subjected to culture, isolation, and biotyping. The antibiogram was carried out via a Kirby-Bauer disk containing eight antibiotics. MRSA was phenotypically confirmed by oxacillin-resistant screening agar base (ORSAB) and genotypically by PCR to detect the presence of the mecA gene. Out of the 400 samples, 47.24% of fresh beef, 37% of contact surfaces, and 64.33% of butchers' knife swabs were Staphylococcus aureus positive. Thirty-two Staphylococcus aureus-positive isolates were confirmed to be MRSA, 50% fresh beef, 28.12% contact surfaces, and 21.87% butcher's knife swabs. MRSA isolates displayed multidrug-resistant traits, with a high resistance of 90.62% against cloxacillin, and a highest susceptibility of 100% to co-trimaxole. The antibiogram showed MRSA strains to be multidrug resistant. Molecular characterisation of the MRSA detected the presence of the mecA gene at a band size of 163bp in all isolates. Strict hygiene of butchers, and working equipment in meat processing and marketing should be of top priority.

Occurrence and Antimicrobial Resistance of Staphylococcus aureus Isolated from Healthy Pet Rabbits.

Background: Staphylococcus aureus is a ubiquitous microorganism and an opportunistic pathogen responsible for numerous diseases in humans and animals, characterized by different clinical pictures with acute or subacute course. S. aureus, due to its great adaptability and versatility in terms of infections and hosts, can be considered a relevant pathogen because of the harmful effects on animal health and its potential for transmission from animals to humans and vice versa. In recent years, a marked increase in multidrug-resistant S. aureus has been reported, posing a serious threat for disease management, food safety, and animal and human health as they limit available therapeutic options. In light of a growing interest of the scientific community for this micro- organism and considering the limited data availability on the prevalence of this pathogen in pet rabbits, the purpose of this research was to evaluate the presence of S. aureus in pet rabbits. Materials and Methods: From November 2021 to December 2022, nasal swabs were collected from 50 pet rabbits from private households in the Campania Region, southern Italy, and underwent analysis for S. aureus detection. Samples were enriched in broth, then inoculated onto nutrient and selective media, including Blood agar base supplemented with 7% sheep blood and Baird-Parker Agar Base, following standard laboratory protocols. Incubations in aerobic conditions at 37°C were performed for 24/48h for colony identification. Antimicrobial susceptibility testing for all S. aureus isolates was conducted using the disc diffusion method. Results: Our results reported the presence of S. aureus in 16/50 (32%) rabbits examined, showing high levels of phenotypic resistance to different antibiotics, in particular penicillin 10U (81.2%) and erythromycin 15 μg (62.5%). Conclusion: The study demonstrated that pet rabbits represent a significant reservoir of S. aureus and contributes to the knowledge on the phenotypic antimicrobial resistance of these bacteria in rabbits raised in a domestic environment.

Isolation, Identification, and Antibiogram of Colistin-resistant Acinetobacter baumannii from Rivers in and around Kathmandu Valley

Background: Acinetobacter baumannii, an opportunistic Gram-negative pathogen, poses an escalating threat in clinical settings due to the rise of multidrug-resistant infections. Despite its clinical significance, there exists a considerable gap in understanding its environmental dissemination.
 Aims and Objectives: The primary objective is to examine the distribution of A. baumannii and its antibiotic resistance in river ecosystems. Specifically, we aim to identify strains resistant to Colistin, a last-resort antibiotic, and elucidate the susceptibility patterns to other antibiotics.
 Materials and Methods: Water samples from 10 rivers were collected and subjected to analysis using Leeds Acinetobacter Agar Base and a series of biochemical tests. Antibiotic susceptibility testing, focusing on Colistin resistance, was performed using standard procedures.
 Results: Out of the 284 isolated strains, 14 (4.9%) exhibited resistance to Colistin, while demonstrating varying susceptibility patterns to other antibiotics. Notably, Gentamycin showed effectiveness against resistant strains (14.28%), while Ceftazidime resistance was complete. Colistin-sensitive strains displayed high susceptibility to Ciprofloxacin (84.44%) and lower susceptibility to Chloramphenicol (53.33%). Carbapenem susceptibility was observed across all isolates.
 Conclusion: The study underscores a concerning environmental presence of multidrug-resistant A. baumannii in rivers around Kathmandu Valley, with Sundarijal being the exception. The findings emphasize the necessity of scrutinizing environmental reservoirs for pathogen spread, advocating for heightened awareness of potential health implications beyond clinical settings. Urgent attention is needed to comprehend and counteract the emergence and dissemination of antibiotic resistance, necessitating comprehensive strategies and continued surveillance

Developing a selective culturing approach for Campylobacter hepaticus.

Campylobacter hepaticus, the causative agent of Spotty Liver Disease (SLD) is an important disease in cage-free egg producing chickens causing mortality and production drops. C. hepaticus is a slow growing Campylobacter easily overgrown by fecal bacteria. It is currently only reliably isolatable from bile samples. A selective media for isolation from feces or environment would assist diagnosis and impact assessment. Growth of five Australian C. hepaticus isolates was studied using Horse blood agar (HBA), sheep blood agar (SBA), Bolton, Preston and Brain Heart Infusion (BHI) base media. Blood and/or bile were added to Bolton, Preston and BHI medias. C. jejuni was used as a positive control. Plates were incubated in duplicate under microaerophilic conditions at 42°C for 10 days and examined at days 3-5 and 7-10 of incubation. Each isolate was examined for sensitivity to 14 antimicrobials using HBA sensitivity plates. Growth was inhibited by BHI and by added bile, while blood improved growth. Further replicates using SBA, HBA, Bolton and Preston media showed best growth on Bolton agar with blood. All five C. hepaticus isolates were resistant to trimethoprim and vancomycin, while four were also resistant to rifampicin and bacitracin. Media based upon Bolton plus blood supplemented with vancomycin and trimethoprim might be used as the most appropriate media for selective growth of C. hepaticus. The addition of bile to media for C. hepaticus isolation and growth will inhibit growth and is not advised.

Kinship analysis of mecA gene of methicillin-resistant Staphylococcus aureus isolated from milk and risk factors from the farmers in Blitar, Indonesia.

There are numerous reports of subclinical mastitis cases in Blitar, which is consistent with the region's high milk production and dairy cattle population. Staphylococcus aureus, which is often the cause of mastitis cases, is widely known because of its multidrug-resistant properties and resistance to β-lactam antibiotic class, especially the methicillin-resistant S. aureus (MRSA) strains. This study aimed to molecular detection and sequence analysis of the mecA gene in milk and farmer's hand swabs to show that dairy cattle are reservoirs of MRSA strains. A total of 113 milk samples and 39 farmers' hand swab samples were collected from a dairy farm for the isolation of S. aureus using Mannitol salt agar. The recovered isolates were further characterized using standard microbiological techniques. Isolates confirmed as S. aureus were tested for sensitivity to antibiotics. Oxacillin Resistance Screening Agar Base testing was used to confirm the presence of MRSA, whereas the mecA gene was detected by polymerase chain reaction and sequencing. A total of 101 samples were confirmed to be S. aureus. There were 2 S. aureus isolates that were multidrug-resistant and 14 S. aureus isolates that were MRSA. The mecA gene was detected in 4/14 (28.6%) phenotypically identified MRSA isolates. Kinship analysis showed identical results between mecA from milk and farmers' hand swabs. No visible nucleotide variation was observed in the two mecA sequences of isolates from Blitar, East Java. The spread of MRSA is a serious problem because the risk of zoonotic transmission can occur not only to people who are close to livestock in the workplace, such as dairy farm workers but also to the wider community through the food chain.

The Ecological Role of Probiotics in in vitro Culture for the Improvement of Health in the Poultry Industry

The general objective of this work was to isolate from yoghourts cultured strains of lactobacilli with potential for use as probiotics in poultry farming. Three yoghourts were cultured to see the presence of lactobacilli in the Rogosa agar base culture medium. It was found that only one yoghourt (number 1) showed the growth of lactobacilli. This yoghourt was immediately selected for further cultivation. Afterwards, the Lactobacillus strains were isolated and fortified under CO2 and then inoculated into a solution of peptone water, which constituted inoculums to be administered to an experimental group of poultry. Another group served as controls. Pathogenic strains of Escherichia coli were also administered to both groups (experimental and control, each comprising five hens). The results showed significant weight gain from the experimental group (positive effect on immunity) and freedom from disease after an incubation period (inhibitory effect of the lactobacillus strains on the Escherichia coli strains), whereas the control group showed less weight gain than the experimental group and development of colibacillosis after an incubation period. The positive effects of the Lactobacillus strains observed on the poultry of the experimental group proved the ecological role of these microorganisms in improving the health of the poultry by inhibiting the effects of pathogenic Escherichia coli strains. This suggests reassuring prospects for the reduction of antibiotic use in both human and animal care.

Deteksi Bakteri Gram-Negatif Pada Permukaan Daun Kemangi (Ocimum basilicum) dari Tiga Pasar Tradisional di Bogor

Ocimum basilicum or basil is commonly consumed raw by Indonesian. However, it is easily contaminated by pathogenic bacteria. This study aimed to detect Gram-negative bacteria in basil leaves obtained from three traditional markets in Bogor. Isolation and quantification of bacteria using serial dilution showed that the average number of bacteria on the surface of basil leaves was 9.6 × 107 CFU/g. Basil leaves obtained from the second traditional market had the highest number of bacteria, namely 11.3 × 107 CFU/g. All isolated bacteria have the same morphology namely bacilli, then further purified and characterized physiologically. The nine bacterial isolates obtained were able to grow on selective-differential media including Salmonella-Shigella agar (SSA), bismuth sulphite agar (BSA), and eosin-methylen blue (EMBA). Gram staining showed that all bacterial isolates were classified as Gram-negative bacteria. The hemolytic ability of bacterial isolates was tested using blood agar base media, three out of nine bacterial isolates were able to produce hemolysin. Molecular identification based on 16S rRNA sequences showed that the bacterial isolates belonged to the Providencia sp., Proteus sp., Pseudomonas sp., and Kluyvera sp. groups.

Research Note: Preliminary results, first detection of Enterococcus cecorum from environmental samples by streaking on X-Gluc containing selective media

The isolation of cultivable E. cecorum from the environment of poultry houses remains a challenge. Environmental samples (dust wipes, equipment swabs, pooled feces) and samples from culled bird vertebras were collected from an infected broiler flock on d 37 posthatching. To isolate the bacterium from the cultivable microbiota, suspensions from the environmental samples were streaked onto a blood agar base medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexylammonium salt (X-Gluc), colistin sulfate, and nalidixin. The chromogenic reaction facilitated the isolation of E. cecorum from contaminated surfaces and pooled feces. Isolates from both the environment and vertebras were confirmed using MALDI-TOF and PCR analysis. Colony appearance and antimicrobial susceptibility tests revealed no phenotypic differences among the isolates. It remained unclear whether the isolates originated from the same clone. However, the principle of isolating the pathogen by streaking on a chromogenic agar may motivate researchers to investigate the transmission routes of infectious isolates, potentially leading to the optimization of biosecurity measures.

Investigating the Occurrence of <i>Escherichia coli</i> O157:H7 in the Final Effluents of Two Wastewater Treatment Plant in the Eastern Cape of South Africa

The final effluents of two wastewater plants located in the Eastern Cape of South Africa were tested for the presence of enterohaemorrhagic Escherichia coli O157:H7 isolates, and characteristics of the isolates obtained were determined. A total of 23 wastewater samples were collected from the treatment plants at the final effluent point after the disinfectant stages of wastewater processing. Altogether, 540 presumptive E. coli isolates were obtained by colony counting on the E. coli O157:H7 chromogenic agar base supplemented with cefixime tellurite and were subcultured onto sorbitol-MacConkey agar and tested for agglutination using the Prolex E. coli O157 latex test reagent kit.The results showed that the 149 suspected colonies from SMAC agar were all negative for the antisera. None of the isolates agglutinated with antisera against E. coli O157 and thus no presence of the bacteria can be confirmed from the treated effluents.The likelihood of the receiving water body and the environment being contaminated with E. coli O157:H7 is therefore minimal. Future monitoring is however recommended

Approaches to the antimicrobial susceptibility testing of Campylobacter to monitor the spread of antibiotic-resistant strains

Introduction. Campylobacteriosis is one of the most common diarrhea-associated infections over the world. The situation is getting worse along with increasing cases of the disease caused by the Campylobacter spp. pathogen resistant to antimicrobials (AMPs). Preventing the disease requires monitoring the spread of resistant Campylobacter strains isolated from both sick people and animals, food, and water. 
 Aim of the study is to evaluate the results of the antimicrobial susceptibility testing of Campylobacter according to the requirements of the EUCAST and CLSI methodologies using Russia-made nutrient media. 
 Materials and methods. Collected and freshly isolated strains of C. jejuni, C. coli, C. fetus and C. lari were used. Campylobacter cultures were subcultured on Campylobacter Agar Base (HiMedia), Preston laboratory-produced medium and iron-erythritol blood agar (Obolensk). Their sensitivity to AMPs was determined by three methods as ffollows: disc diffusion, gradient diffusion and microdilutions using Mueller-Hinton agar, and broth of two manufacturers (BD BBL and Obolensk) according to EUCAST and CLSI.
 Results. Using Russian-made and imported Mueller-Hinton agar and broth allowed obtaining identical results for AMPs susceptibility of Campylobacter spp. The campylobacter strains were attributed to the same susceptibility category by all three methods in frame of any methodology (EUCAST or CLSI) when interpreting results. Due to differences in cutoffs of MIC and inhibition zone diameters in the EUCAST and CLSI standards, there were some differences in the interpretation of the results.
 Limitations. Eight strains of four species Campylobacter were tested for their susceptibility to three antimicrobials by three methods according to EUCAST and CLSI methodology.
 Conclusion. The results obtained confirm the possibility of applying a complex of domestic nutrient media for cultivating and monitoring the spread of antibiotic resistant strains of Campylobacter spp. This is especially important in view of implementing the import substitution program.

Methicillin Resistant Staphylococcus aureus (MRSA) Isolation and mecA Gene Detection from Milk and Farmer Hand Swab in Tulungagung, Indonesia

Staphylococcus aureus is a harmful bacterium that often contaminates milk; hence it is believed to become a severe health risk for humans . S. aureus resistant to β‑lactam drugs can be termed methicillin resistant Staphylococcus aureus (MRSA). Dairy farms have a high incidence of MRSA infections due to the repeated use of the same medicines on dairy cows and the physical contact between farmers and cows during milking. This study looked for MRSA in dairy cow milk and farmer hand swabs in Tulungagung, Indonesia. Using oxacillin and cefoxitin diffusion disks, phenotypic detection approaches were evaluated, then transferred to the Oxacillin Resistance Screening Agar Base (ORSAB) test and genotypically verified using PCR to find the mec A gene encoding MRSA. One hundred ten dairy cow milk samples and 45 farmer's hand swabs were collected from Tulungagung, East Java, Indonesia. Mannitol salt agar (MSA) was used for cultivation and purification. The disk-diffusion test used oxacillin and cefoxitin to identify S. aureus resistance. Oxacillin and cefoxitin-resistant S. aureus isolates were tested for MRSA using ORSAB. In addition, MRSA isolates were PCR-tested for the mec A gene. S. aureus was found in 110 (70.97%) of 155 isolates. Of the total 110 isolates of S. aureus , 16 (14.54%) and 39 (35.45%) were known to be resistant to Cefoxitin and Oxacillin, respectively. When tested with ORSAB, 23 isolates from 55 resistant isolates showed positive results for MRSA. Dairy milk was the source of most MRSA which is 15 isolates, while hand swabs only carried 8 isolates. However, PCR analysis only found mec A gene in two isolates. According to this study, many MRSA isolates were found from dairy farms in Tulungagung, Indonesia, but only a few have the mecA gene.

Influence of the storage temperature of raw sheep milk on the spoilage potential of Pseudomonas spp.

Due to their quality, high yield and nutritional value, sheep dairy products have been gaining market space and attracting new producers. Pseudomonas spp. is the most common psychrotrophic genus present in bovine refrigerated raw milk and is characterized by intense lipoproteolytic activity. Metalloprotease aprX is one of the enzymes produced by Pseudomonas spp. which has great industrial importance, since it has specific activity in the deterioration of casein, resulting in significant changes in physicochemical properties and sensorial characteristics of raw milk and dairy products. The aim of this study was to observe the occurrence of Pseudomonas spp. in raw sheep milk and evaluate the influence of storage temperature on the spoilage potential of the strains. Raw sheep milk samples were stored at 4 °C or 9 °C (72 h) and plated in Pseudomonas Agar Base supplemented with CFC (25 ºC / 48 h). The isolates characteristic of Pseudomonas had their genus, species (P. putida, P. fluorescens and P. aeruginosa), and presence of gene aprX confirmed by Polymerase Chain Reaction (PCR). The lipoproteolytic capacity and the spoilage potential of the isolates were evaluated in Milk Agar (10%) and Tributyrin Agar Base (1%) (21 °C / 72 h). Initial (2 h) populations of Pseudomonas spp. ranged from 1.0 to 4.2 log CFU/mL; after 72 h, the values observed in milk samples stored at 9 °C (3.0–7.0 log CFU/mL) were higher than those obtained in samples at 4 °C (1.6–6.0 log CFU/mL). A total of 255 strains of Pseudomonas spp. were isolated: 30.2% (77) from milk stored at 4 °C and 69.8% (178) from milk at 9 °C. At both temperatures, P. fluorescens was predominant, followed by P. putida. P. aeruginosa strains, isolated only from samples stored at 9 °C, showed high lipoproteolytic capacity and were characterized by higher proteolytic potential than lipolytic. P. fluorescens was the only species to present high proteolytic (halos > 2 cm) potential. Considering a single storage temperature (4 °C and 9 °C), only P. fluorescens showed higher proteolytic potential than lipolytic. The aprX gene was predominant in P. fluorescens and P. putida isolates (4 °C and 9 °C); strains obtained from milk stored at 4 °C showed higher proteolytic potential than those isolated from milk at 9 °C. All strains of P. fluorescens and only 46.42% of P. putida presented proteolytic capacity at 9 °C. The aprX gene was observed in all P. aeruginosa strains, but only 95.65% of them showed proteolytic capacity. The lipolytic potentials of P. fluorescens and P. putida were not influenced by milk storage temperature. P. fluorescens was the only species to present high lipolytic potential. P. putida isolated from milk at 9 °C was the only species to present higher lipolytic potential than proteolytic. According to the microbiota and the storage temperature, there may be a predominance of proteolytic or lipolytic enzymes in milk and consequently, defects in sheep dairy products. It should also be considered that most of the isolates of Pseudomonas spp. presented lipoproteolytic capacity, which emphasizes the importance of low initial populations of this microorganism in raw sheep-chilled milk, mainly when used in the production of dairy products.

Evaluation of Cefoxitin Disc Diffusion and Chromogenic Agar in the Detection of Methicillin Resistant Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) has a worldwide distribution and is an important cause of clinical and epidemiological problems. The aim of this study was to evaluate the usefulness of some phenotypic methods for the detection of methicillin resistant S. aureus in clinical laboratories. The study is cross sectional. A total of 93 S. aureus isolates were tested using cefoxitin disk diffusion (CDD) and oxacillin resistance screening agar base (ORSAB) with reference to mecA gene PCR. Of the 93 isolates, CDD test showed 34 were MRSA, while ORSAB recorded 42. MecA gene was detected by PCR in 34 of the isolates. The CDD showed 97.1% sensitivity and 98.3% specificity and therefore superior to ORSAB with sensitivity 97.1% and specificity 84.7%. The cefoxitin disk test required no special test conditions and can improve the reliability of routine tests for the detection of MRSA. CDD test can thus be used as a cheap and reliable alternative to PCR for the detection of MRSA in resources limited settings.