Presumptive Colonies Research Articles

UV-light fluorescence as a confirmation method for presumptive Legionella colonies isolated from water samples.

Legionnaires' disease (LD) is a severe and potentially fatal form of bacterial pneumonia caused by Legionella spp. We evaluated the use of UV-light for detecting Legionella non-pneumophila in water samples according to the NEN-EN-ISO 11731:2017 methodology (reference method) in a collaborative effort involving 10 laboratories. First, a test panel was constructed of 298 strains: 157 Legionella strains and 141 non-Legionella strains were cultured on buffered charcoal yeast extract (BCYE)-medium and confirmed according to ISO 11731: 2017 (cultured on BCYE agar plates with and without l-cysteine), and by matrix-assisted laser desorption-ionization time of flight or next generation sequencing. All strains were additionally exposed to an UV-light to assess if they showed a bright blue fluorescence effect (UV-positive) or not (UV-negative). Second, in an interlaboratory study, 10 laboratories analyzed a blinded set of 16 Legionella strains and 8 non-Legionella strains using both methods. The test panel analyses showed 100% accordance between the UV-light and reference method. In addition, the interlaboratory study results showed full agreement between both methods. Our results support the implementation of UV-light detection to confirm Legionella presumptive colonies during analyses of water samples according to the NEN-EN-ISO 11731:2017 methodology. Implementation of UV-light confirmation could reduce workload, time-to-result and costs for the analyses of water samples for the presence of Legionella.

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 of Helicobacter pullorum in Retail Chicken Meat: A One-Health Approach to Consumer Health Protection.

Helicobacter pullorum is an emerging foodborne pathogen that commonly colonizes the gastrointestinal tract of poultry, causing gastroenteritis. It has been related to several clinically important infections, including colitis and hepatitis, inflammatory bowel disease, recurrent diarrhea, and bacteremia in the human population. The bacterium may be transmitted to humans through undercooked poultry meat. In order to investigate the occurrence of H. pullorum in raw retail chicken meat (thighs and breasts), we analyzed 240 samples: 120 chicken thigh and 120 chicken breast samples. The samples were analyzed by means of an isolation protocol using Steele and McDermott's modified filtration technique on Brucella agar supplemented with 5% of defibrinated sheep's blood. The presumptive colonies were biochemically identified and analyzed using a previously described conventional PCR test based on the 16S rRNA gene. In total, 35% of analyzed samples were positive using the microbiological protocol and 45% were positive by PCR. These results suggest that H. pullorum can be transmitted to humans through the handling and consumption of raw poultry meat, representing a risk for food business operators and consumers. Efforts to control H. pullorum in broiler meat should prioritize the implementation of stringent hygienic practices across all stages of the food chain, from the farm to the consumer.

Dissemination of mcr-1 and β-lactamase genes among Pseudomonas aeruginosa: molecular characterization of MDR strains in broiler chicks and dead-in-shell chicks infections

ObjectivesPseudomonas aeruginosa (P. aeruginosa) is one of the most serious pathogens implicated in antimicrobial resistance, and it has been identified as an ESKAPE along with other extremely significant multidrug resistance pathogens. The present study was carried out to explore prevalence, antibiotic susceptibility phenotypes, virulence-associated genes, integron (int1), colistin (mcr-1), and β-lactamase resistance' genes (ESBls), as well as biofilm profiling of P. aeruginosa isolated from broiler chicks and dead in-shell chicks.DesignA total of 300 samples from broiler chicks (n = 200) and dead in-shell chicks (n = 100) collected from different farms and hatcheries located at Mansoura, Dakahlia Governorate, Egypt were included in this study. Bacteriological examination was performed by cultivation of the samples on the surface of both Cetrimide and MacConkey’s agar. Presumptive colonies were then subjected to biochemical tests and Polymerase Chain Reaction (PCR) targeting 16S rRNA. The recovered isolates were tested for the presence of three selected virulence-associated genes (lasB, toxA, and exoS). Furthermore, the retrieved isolates were subjected to phenotypic antimicrobial susceptibility testing by Kirby–Bauer disc diffusion method as well as phenotypic detection of ESBLs by both Double Disc Synergy Test (DDST) and the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). P. aeruginosa isolates were then tested for the presence of antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, OXA-2, VEB-1, SHV, TEM, and CTX-M). Additionally, biofilm production was examined by the Tube Adherent method (TA) and Microtiter Plate assay (MTP).ResultsFifty –five isolates were confirmed to be P. aeruginosa, including 35 isolates from broiler chicks and 20 isolates from dead in-shell chicks. The three tested virulence genes (lasB, toxA, and exoS) were detected in all isolates. Antibiogram results showed complete resistance against penicillin, amoxicillin, ceftriaxone, ceftazidime, streptomycin, erythromycin, spectinomycin, and doxycycline, while a higher sensitivity was observed against meropenem, imipenem, colistin sulfate, ciprofloxacin, and gentamicin. ESBL production was confirmed in 12 (21.8%) and 15 (27.3%) isolates by DDST and PCDDT, respectively. Antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, SHV, TEM, and CTX-M), were detected in 87.3%, 18.2%, 16.4%, 69.1%, 72.7%, and 54.5% of the examined isolates respectively, whereas no isolate harbored the OXA-2 or VEB-1 genes. Based on the results of both methods used for detection of biofilm formation, Kappa statistics [kappa 0.324] revealed a poor agreement between both methods.Conclusionsthe emergence of mcr-1 and its coexistence with other resistance genes such as β-lactamase genes, particularly blaOXA-10, for the first time in P. aeruginosa from young broiler chicks and dead in-shell chicks in Egypt pose a risk not only to the poultry industry but also to public health.Graphical

The Pervasiveness of Bio-Hazards in Street-Vended Dairy Products

A total of 60 diverse dairy product samples, including ice cream, Ras cheese, and Kariesh cheese (20 of each), were randomly collected from street vendors in different regions of Alexandria governorate, Egypt to investigate the prevalence of pathogenic E. coli. The outcomes revealed presumptive positive colonies of 83.33% and 80% in ice cream and Kariesh cheese samples, respectively. Confirmatory tests via conventional biochemical identification affirmed the presence of E. coli in 80% of the samples. However, Vitek 2 compact testing revealed that only 46.66% were positive, in contrast to PCR techniques, which substantiated a prevalence of E. coli in only 25% of the samples, with 35% in street-vended Ras cheese and 25% in Kariesh cheese. The phoA gene, integral to E. coli, was prominently featured in street-vended Ras cheese samples at a striking 100%, while the virulence genes stx1 and stx2 were conspicuously absent, except for sporadic instances of the eaeA gene in street-vended products. These findings uncovered a nuanced landscape of bacterial presence, with E. coli exhibiting a significant prevalence disparity among product sources, particularly with street-vended items showing markedly high contamination rates. It is advised that awareness programs should be established to reduce dairy product contamination among street vendors. Keywords: Dairy; Street-vended; Escherichia coli; Contamination.

Extensively drug-, ciprofloxacin-, cefotaxime-, and azithromycin-resistant Salmonella enterica serovars isolated from camel meat in Egypt

Given the great importance of Salmonella as a leading foodborne pathogen of global concern and the few available data regarding its prevalence in camel meat, the present study aimed to determine the prevalence, antimicrobial resistance (AMR) profile, virulence genes, β-lactamase genes of Salmonella enterica serovars isolated from camel meat marketed in Egypt. Forty-five (29.6 %) of the 152 camel meat samples examined were positive for Salmonella spp. Among the 432 Salmonella presumptive colonies isolated, 128 were molecularly verified as Salmonella after confirmation by PCR targeting the Salmonella marker (invA) gene. Virulence genes, encompassing stn, spvC, and hilA genes, were detected in 91.4 % (117/128), 20.3 % (26/128), and 80.5 % (103/128) of the isolates, respectively. S. Enteritidis, S. Typhimurium, S. Cerro, and S. Montevideo were the most prevalent serovars with incidences of 25 % (32/128), 15.6 % (20/128), 15.6 % (20/128), and 12.5 % (16/128), respectively. Interestingly, 56.3 %, 53.1 %, 37.5 %, 28.1 %, 21.9 %, 18.8 %, 12.5 %, and 3.1 % of the isolates tested showed resistance to cefepime, ciprofloxacin, levofloxacin, cefotaxime, gentamicin, colistin, meropenem, and azithromycin, respectively. Salmonella isolates showed resistance to at least one antibiotic, with a mean multiple antibiotic resistance (MAR) index of 0.472. Interestingly, 59.4 %, 15.6 %, and 3.1 % of the isolates were categorized into multidrug-resistant, extensively drug-resistant, and pan-drug-resistant, respectively. Only 23 (25 %) of the 92 ampicillin-resistant isolates were proven to be Extended Spectrum Beta-Lactamase (ESBL)-producing Salmonella, in which β-lactamase (bla) genes were detected. The blaOXA-2 was the most existing gene where it was detected lonely in 10 of the ampicillin-resistant isolates and coexisted with blaCMY-1 in 4 isolates and with blaCMY-2 in a single isolate. The blaCMY-1, however, existed in 11 isolates, whereas the blaCMY-2 gene was only detected in 3 isolates tested. The present findings affirm that camel meat could be a leading reservoir for multi- and extensively-drug-resistance β-lactamase-producing Salmonella, representing a global public health challenge. Therefore, further research is necessary to detect the prevalence and AMR of Salmonella serovars from camel meat in Egypt and other countries to put camel meat as a source of Salmonella in foods of animal origin.

Occurrence of Vibrio spp. in Selected Recreational Water Bodies in Belgium during 2021 Bathing Season.

In recent years, a global increase in the number of reports of human vibriosis involving V. cholerae non-O1/O139 (NOVC) and other Vibrio spp. has been observed. In this context, the Belgian National Reference Center for Vibrio conducted an assessment of the presence of Vibrio spp. in recreational waters. Water sampling was performed monthly in different lakes in Wallonia and Flanders, including the North Sea. The collected water was then filtrated and cultured, and Vibrio spp. was quantified according to the Most Probable Number (MPN). Presumptive colonies were confirmed via MALDI-TOF, and PCR for virulence genes was applied if justified. No Vibrio spp. was found in the analyzed water bodies in Wallonia. However, NOVC was isolated from three different lakes in Flanders and from coastal water. In addition, V. alginolyticus and V. parahaemolyticus were also detected in coastal water. No clear impact of the pH and temperature was observed on Vibrio spp. occurrence. Our study demonstrates the presence of Vibrio spp. in different bathing water bodies, mostly in the north of Belgium, and supports the recommendation to include Vibrio spp. as a water quality indicator for bathing water quality assessment to ensure the safety of water recreational users in Belgium.

Potential probiotic characterization of lactic acid bacteria isolated from Duimaj, an Iranian traditional snack food, using biochemical, molecular and computational approaches

The aim of this study was isolation and identification of lactic acid bacteria from Duimaj (an Iranian traditional snack food) and in vitro characterization of probiotic features. In total, thirty presumptive LAB colonies were surveyed, of which 22 exhibited high rates of survival in artificial gastrointestinal fluids, which were found to belong to Lactobacillus, Enterococcus, and Pediococcus genera based on 16 S-rRNA gene sequence. The selected strains had strong antagonistic effects, with auto-aggregation abilities ranging from 26.52 to 81.50% and co-aggregation capacity up to 88.33 and 80.14% with E. coli and S. aureus, respectively. Moreover, the cell surface hydrophobicity of strains ranged from 31.06 to 85.62%. Results indicated that nine isolated LAB harbored the cholesterol assimilation rates greater than 30%. Moreover, none of the selected strains showed hemolytic activity. Among the isolates, five strains were negative for all nine tested virulent genes, and exhibited favorable adhesion percentage to human intestinal epithelial cells ranging from 31.76 to 52.37. Hence, naturally occurring Lactiplantibacillus plantarum and Lactiplantibacillus pentosus isolated from Duimaj were proposed as suitable probiotic candidates for use in functional foods.

Viable Mycobacterium avium subsp. paratuberculosis Colonizes Peripheral Blood of Inflammatory Bowel Disease Patients.

Pathobionts, particularly Mycobacterium avium subsp. paratuberculosis (MAP) and Escherichia coli isolates with adherence/invasive ability (AIEC) have been associated with inflammatory bowel disease (IBD), particularly Crohn's disease (CD). This study aimed to evaluate the frequency of viable MAP and AIEC in a cohort of IBD patients. As such, MAP and E. coli cultures were established from faecal and blood samples (with a total n = 62 for each) of patients with CD (n = 18), ulcerative colitis (UC, n = 15), or liver cirrhosis (n = 7), as well as from healthy controls (HC, n = 22). Presumptive positive cultures were tested by polymerase chain reaction (PCR), for a positive confirmation of MAP or E. coli identity. E. coli-confirmed isolates were then tested for AIEC identity using adherence and invasion assays in the epithelial cell line of Caco-2 and survival and replication assays in the macrophage cell line of J774. MAP sub-culture and genome sequencing were also performed. MAP was more frequently cultured from the blood and faecal samples of patients with CD and cirrhosis. E. coli presumptive colonies were isolated from the faecal samples of most individuals, in contrast to what was registered for the blood samples. Additionally, from the confirmed E. coli isolates, only three had an AIEC-like phenotype (i.e., one CD patient and two UC patients). This study confirmed the association between MAP and CD; however, it did not find a strong association between the presence of AIEC and CD. It may be hypothesized that the presence of viable MAP in the bloodstream of CD patients contributes to disease reactivation.

Levels and Distribution of Salmonella in Naturally Contaminated Cashews

Raw materials associated with foodborne illness outbreaks are rarely available for evaluation. The levels and distribution of Salmonella were determined in naturally contaminated raw cashews linked to a salmonellosis outbreak associated with a fermented cashew cheese analog. Two unopened 22.7-kg boxes from a single lot of cashew kernel pieces were each divided into seven approximately equal units, 14 in total. Three 10-g subsamples per unit (n = 21) were evaluated for aerobic plate count (APC), coliform counts, and Escherichia coli counts, and 10 50-g subsamples per unit (n = 70) were enriched for the presence of Salmonella. Presumptive Salmonella-positive colonies were confirmed using CHROMagar Salmonella and real-time PCR (invA) and then serotyped using antigenic methods and genome sequencing prediction tools. APC and coliform counts ranged from 1.81 to 5.47 (mean 2.44 ± 0.63) log CFU/g and 0.60 to 5.20 (mean 1.74 ± 0.80) log CFU/g, respectively. Salmonella was recovered from four units in Box 1 and all seven units in Box 2. One of the 10 subsamples was positive in all but four of the positive units; one (Box 1) and three (Box 2) units had two positive subsamples. The level of Salmonella in the two boxes combined was 0.0023 most probable number/g (95% confidence interval [0.0014, 0.0038]). Salmonella Urbana was isolated from three of five positive subsamples in Box 1 and eight of 10 positive subsamples in Box 2. Salmonella Fresno and Vinohrady were unique to single subsamples from Box 1, and Salmonella Nima was isolated from two subsamples from Box 2. Of the four serovars recovered, Salmonella Urbana and Salmonella Vinohrady were in common with outbreak-associated clinical or product isolates. Understanding the distribution and concentration of Salmonella in naturally contaminated cashews provides important information for hazard analysis and risk assessments for soaked and fermented cashew products.

Confirmation of presumptive Legionella colonies on culture media according to ISO 11731:2017: principles, problems, and practice.

The ISO 11731 norm, published in 2017, describes a method to identify and enumerate Legionella based exclusively on the confirmation of presumptive colonies by subculturing them on BCYE and BCYE-cys agar (BCYE agar without L-cysteine). Despite this recommendation, our laboratory has kept confirming all presumptive Legionella colonies by combining the subculture method with the latex agglutination and polymerase chain reaction (PCR) assays. Here, we show that the ISO 11731:2017 method adequately performs in our laboratory according to ISO 13843:2017. We compared the performance of the ISO method in detecting Legionella in typical and atypical colonies (n=7156) from health care facilities (HCFs) water samples to that of our combined protocol, and we found a 2.1% false positive rate (FPR), underscoring the importance of combining agglutination test and PCR with subculture to achieve optimal confirmation. Lastly, we estimated the water system disinfection cost for HCFs (n=7), which due to false positive results, would display Legionella values exceeding the threshold of risk acceptance established by the Italian guidelines. Overall, this large-scale study indicates that the ISO 11731:2017 confirmation method is error-prone, leading to significant FPRs, and higher costs for HCFs due to remedial actions on their water systems.

Rapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 by mass spectrometry directly from the isolate, using 10 potential biomarker peaks and machine learning predictive models.

Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157 : H7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157 : H7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3 % (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157 : H7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157 : H7Conclusion. Given that STEC O157 : H7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5 % and specificity=100.0 %), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157 : H7 versus other diarrhoeagenic E. coli in the future.

Molecular Characterization of Enterococcus faecalis and Enterococcus faecium Isolated from a Meat Source in Shahrekord Local Markets, Iran.

Enterococcus faecalis (E. faecalis) and E. faecium (E. faecium) are commensals of the gastrointestinal biota of humans and animals and are considered opportunistic pathogens. This study aimed to improve the knowledge about E. faecalis and E. faecium isolated from meat. To this end, 104 meat samples were collected from sheep, goats, cattle, and calves from Shahrekord local markets in Iran. Presumptive colonies of E. faecalis and E. faecium were isolated from the samples and subjected to identification tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was performed to detect some virulence genes. Furthermore, randomly amplified polymorphic DNA typing and repetitive element sequence-based PCR fingerprinting were used to show the clonal relatedness of the isolates. The results revealed that enterococci were present in 90 out of 104 samples. Specifically, E. faecalis and E. faecium were the commonly isolated species, with the predominance of E. faecalis, which exhibited high resistance to streptomycin (95%) but was susceptible to vancomycin (85.6%). Virulence genes detection showed that ccf and cpd genes were the most prevalent genes in both species. In addition, the molecular typing method indicated that the isolates belonged to separate subgroups. This study shows the contamination of meat products by potential pathogens and resistant enterococci. There is a need to implement regular surveillance to monitor the emergence of antimicrobial-resistant E. faecalis and E. faeciumin food, particularly in meat production.

Toward the Adoption of Loop-Mediated Isothermal Amplification for Salmonella Screening at the National Antimicrobial Resistance Monitoring System's Retail Meat Sites.

The National Antimicrobial Resistance Monitoring System (NARMS) is a One Health program in the United States that collects data on antimicrobial resistance in enteric bacteria from humans, animals, and the environment. Salmonella is a major pathogen tracked by the NARMS retail meat arm but currently lacks a uniform screening method. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the rapid screening of Salmonella from 69 NARMS retail meat and poultry samples. All samples were processed side by side for culture isolation using two protocols, one from NARMS and the other one described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Overall, 10 (14.5%) samples screened positive by the Salmonella LAMP assay. Of those, six were culture-confirmed by the NARMS protocol and six by the BAM method with overlap on four samples. No Salmonella isolates were recovered from samples that screened negative with LAMP. These results suggested 100% sensitivity for LAMP in reference to culture. Antimicrobial susceptibility testing and whole-genome sequencing analysis confirmed identities of these isolates. Using the BAM protocol, all Salmonella isolates were recovered from samples undergoing Rappaport-Vassiliadis medium selective enrichment and presumptive colonies (n = 130) were dominated by Hafnia alvei (44.6%), Proteus mirabilis (22.3%), and Morganella morganii (9.9%) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method comparison study clearly demonstrated the benefit of a rapid, robust, and highly sensitive molecular screening method in streamlining the laboratory workflow. Fourteen NARMS retail meat sites further verified the performance of this assay using a portion of their routine samples, reporting an overall specificity of 98.8% and sensitivity of 90%. As of July 2022, the vast majority of NARMS retail meat sites have adopted the Salmonella LAMP assay for rapid screening of Salmonella in all samples.

Occurrence of community-acquired Panton-Valentine leukocidin-producing and enterotoxin-producing methicillin-resistant staphylococci in companion dogs

In Nigeria, available data on drug-resistant bacterial infections that are caused by companion dogs are scarce. Hence the present study evaluated the occurrence of some community-acquired toxigenic methicillin-resistant staphylococci (MRS) on companion dogs harboured in Nigerian homes, as a pointer to the extent of exposure of humans to these pathogens. Samples were collected from 70 healthy companion dogs during dry and rainy season periods by swabbing a 125 cm2 fur area on the lumbar and thoracic sites. Phenotypic tests, Kirby Bauer disc diffusion test and 16S rRNA gene analysis were used to identify presumptive colonies of staphylococci and MRS. Molecular methods were employed to detect Panton-Valentine leukocidin (PVL) and prototypic enterotoxin B in MRS isolates. The counts of staphylococci on fur of companion dogs during the rainy season exceeded usual limits of bacteria (≤ 2.54 log10 CFU cm-2) on a healthy dog, thus, suggesting that companion dogs harboured in homes situated in Nigeria may be reservoirs of bacteria, especially during rainy season. The mean counts of staphylococci during the rainy season were estimated at 3.09 ± 2.78 log10 CFU cm-2 and 2.77 ± 2.43 log10 CFU cm-2 in Edo and Delta States, respectively. The main Staphylococcus species that were carried on fur of companion dogs included S. pseudintermedius, S. aureus, S. epidermidis, S. simulans and S. saprophyticus. Amongst the staphylococci, expression of methicillin and multidrug resistance was mainly exhibited by S. pseudintermedius and S. aureus, while enterotoxigenicity was mainly expressed by methicillin-resistant S. aureus. Enterotoxigenic S. aureus was carried on the fur of companion dogs during the rainy season at estimated prevalence of 8.57% in both Edo and Delta States, respectively; while PVL-producing S. aureus was estimated at 5.71% and 2.86%, with PVL-producing S. pseudintermedius estimated at 25.71% and 34.29%, respectively. The high prevalence of toxigenic-producing isolates seen on the fur of companion dogs, especially during rainy season, could pose a risk for humans, particularly those that harbour pet dogs at their homes.

Prevalence and antimicrobial susceptibility pattern of Campylobacter jejuni in raw retail chicken meat in Metropolitan Accra, Ghana

Although, Campylobacter spp. are a major cause of foodborne gastroenteritis, its occurrence and antimicrobial resistance traits have not been well defined in low income countries, particularly in Africa. In this study, retail chicken was sampled (n = 400) between February 2019 to January 2020 in Metropolitan Accra, Ghana, to determine the prevalence and antimicrobial susceptibility pattern of Campylobacter jejuni. Raw chicken samples were obtained in wet markets (n = 315) and supermarkets (n = 85) and each subjected to direct plating and broth enrichment according to standard culture methods for Campylobacter spp. with the identity of presumptive positive colonies confirmed by MALDI-TOF. The susceptibility of isolates to antibiotics commonly used for campylobacteriosis in humans (in order to reflect the One Health significance of Campylobacter at the human-food interface) were then assessed by disc diffusion. A prevalence of 38.3% was recorded and all isolates were confirmed as Campylobacter jejuni. Enrichment yielded 127 positives while direct plating yielded 55 positives with low level of agreement in detection between these assays (Kappa = 0.15). Among samples positive by direct plating, the mean Campylobacter count was 1.9 log10 CFU/g (sd ±0.8). About 13% (7/55) of the samples positive by direct plating contained counts of 3log and above. Samples from the wet market yielded more positives than those from the supermarket with the rate of isolation from wet markets being 1.6 times that of the supermarket. Among 182 isolates characterized for their antimicrobial susceptibility, resistance to fluoroquinolones was 99.5%, tetracyclines 100% and macrolides 26.9%. Multi-drug resistance was also observed in 26.9% of the screened isolates. The findings point to a potential high level of exposure of humans to Campylobacter jejuni through chicken meat and thus the need for education on hygienic preparation and handling of raw chicken. High rates of resistance to classes of antimicrobials critically important for treating Campylobacter infections in humans; fluroquinolones and macrolides, affirm the need for stronger regulatory control of antimicrobials and enhanced antimicrobial stewardship in chicken production.

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

Prevalence and antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus isolated from bovine raw milk in Njombe region, Tanzania

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) creates a serious public health concern due to its ability to colonize and infect humans and animals. This cross-sectional study investigated the prevalence, antibiotic susceptibility pattern of S. aureus and MRSA isolated from bovine raw milk in the Njombe region, Tanzania. A total of 470 samples, including 389 raw milk samples collected at farm level, 57 raw milk samples from bulk milk-can at collection centers, and 24 swab samples from bulk milk cans. The samples were cultured on mannitol salt agar, presumptive colonies were sub-cultured onto blood agar for the isolation of S. aureus which was subsequently preliminarily confirmed using microbiological and biochemical tests. Further, confirmation of isolates was done using conventional PCR targeting gltB gene for S. aureus and mecA gene for MRSA which was later sequenced. Isolates were tested for antibiotic susceptibility by using the disc agar diffusion method. The overall prevalence of S. aureus in the study was 22.6\% (106/470), with 2.9\% (14/470) being MRSA. Both S. aureus and MRSA showed high resistance to penicillin (74\%, 8.5\%) and ampicillin (78\%, 11.3\%), respectively. A total of 81 (77\%) isolates were resistant to at least one antibiotic and 14 isolates (13.2\%) showed multidrug-resistant (MDR); with frequent antibiotic resistance patterns being to cefoxitin, penicillin, ciprofloxacin, tetracycline, and erythromycin. In conclusion, the prevalence and the MDR patterns exhibited by S. aureus and MRSA observed in this study provide baseline data for planning mitigation measures to safeguard public health.

Using Whole Genome Sequencing to Investigate a Mock-Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in Real-Time.

Healthcare associated infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major concern in Portuguese hospitals. Whole genome sequencing (WGS) can improve infection control, but this practice is not routinely used by hospital clinical laboratories in Portugal. We simulated the investigation of a CRKP outbreak based on WGS, with the aim of determining, in the minimum possible time, genetic relatedness between CRKP clinical and environmental isolates. Ten CRKP clinical isolates routinely obtained in the hospital laboratory were used. Forty environmental samples - from sinks and sink drains of ward rooms - were collected. Environmental samples were plated on selective media and presumptive CRKP colonies were isolated. Total DNA was extracted from all putative CRKP isolates and sequenced. Clonal relatedness was determined by multi-locus sequence typing and core genome single nucleotide polymorphism analysis; the presence of carbapenemase genes was evaluated. Clinical isolates were characterized in 48 hours: eight strains were confirmed as CRKP, of which six were of ST13 and carried blaKPC-3. Environmental samples results were obtained in six days: eight CRKP were isolated from which five were of ST13 and carried blaKPC-3. Clinical and environmental ST13 isolates were highly related: ten (of 11) isolates differed from each other in < 0.001% of 2 172 367 core nucleotides. WGS can be used as a high-resolution effective tool to investigate healthcare associated infections and track routes of dissemination in real-time. In Portugal, routine use of WGS to improve infection control could thrive through collaborative initiatives between hospitals and research institutes.

Characterization of Faecal Enterococci from Wild Birds in Turkey and Its Importance in Antimicrobial Resistance

This research aimed to investigate the diversity of faecal enterococci isolated from wild birds, to detecttheir antibiotic resistance patterns and to determine their distribution of genes related to vancomycin resistance. Additionally, to investigate their virulence factors that are important in the development of the disease. One hundred seven cloacal/rectal samples were inoculated onto Enterococcus Agar, and presumptive colonies were identified and confirmed by PCR. Multiplex PCR assays were used to screen vanA, vanB, vanC1 and vanC2/3. The virulence-related genes; ace, gelE, efa and agg were determined by PCR. Among the 103 enterococci, 62 E.faecalis, 23 E.faecium 3 E.gallinarum, 2 E.durans, 1 E.casseliflavus and 12 Enterococcus spp. were identified. Of the 103 enterococci, 26 were found to be resistant against to three or more antibiotics. The highest percentages were detected for chloramphenicol (52%), tetracycline (33%) and erythromycin (30%). Two E.gallinarum isolates were harboring three virulence factors, and one isolate was carrying a single virulence factor. There is no virulence factor in the E.casseliflavus isolate. Also, vanA and vanB genes were not found. Forty-two of 103 enterococci were harboring virulence factors, more frequently in E.faecalis. Forty-two enterococci carried efa A, 31 isolates carried gel E, and ace was found in 18 isolates. Virulence gene agg was not detected. When the results of the study were evaluated in general, multiple drug resistance was described as 25%. Considering the risk of polluting the water resources of wild animals, it is suggested that the continuity of this type of epidemiological study in wildlife animals is necessary. In conclusion, the wild birds may act as substantial reservoirs carrying antimicrobial resistance among enterococci and estimate the potential risk for man, pets and farm animals.