Antimicrobial resistance characteristics of Extended Spectrum Beta Lactamase (ESBL)- producing Escherichia coli from dairy farms in the Sleman district of Yogyakarta province, Indonesia

Extended Spectrum Beta Lactamase (ESBL)-producing Escherichia coli (E. coli) infections are a global health challenge resulting from human contact with infected animals and contaminated farm environments. This study aims to identify antimicrobial resistance patterns of ESBL-producing E. coli isolated from dairy farms in the Sleman District of Yogyakarta Province, Indonesia. Ninety-three dairy farms with a history of antibiotic use in the previous 6 months were identified. Samples were collected from 6 different sources (feces, milk, wastewater, animal drinking water, feed and rinses of workers’ hands) on each farm during August through November 2020. These samples were cultured with conventional microbiological methods for the isolation of ESBL-producing E. coli. ESBL-producing E. coli was identified in one or more of the sources in 54% (50/93) of the dairy farms sampled. Fecal samples were the most commonly positive (25%) while wastewater, animal drinking water feed, milk and hand rinses were positive at 16%, 10%, 5%, 4% and 3% respectively. Colonies from each positive sample were screened for antibiotic susceptibility test using the Vitek-2 system. Resistance to trimethoprim/sulfamethoxazole, tetracycline and gentamicin were found in 74%, 63% and 48% of the isolates, respectively. Multidrug resistant (MDR) was identified in 50% (63/127) of the isolates. In conclusion, ESBL-producing E. coli appears widespread in dairy farms using antibiotics and antimicrobial resistance among these bacteria is common in this study area. Further study of the risk of human transmission from contaminated cattle and their environments could benefit the national antimicrobial resistance strategic plans

Labor- und Praxisversuche zur Reduktion der Salmonellenkontamination von rohen Hühnerbrustfilets

Experimental Reduction of Salmonella in Raw Chicken Breasts The objective of this study was to identify a suitable decontamination method for chicken meat. For this, investigations on a combination of physical and chemical interventions were carried out in the laboratory, and further experiments were done in the process-line of a poultry meat producing enterprise. Initial trials determined the appropriate temperature, contact time and treatment solution for the immersion treatments which are based on sensory attributes such as the surface appearance, taste, odor and texture of the chicken meat. The antimicrobial effect of bacterial reduction in pure culture and of artificially contaminated chicken meat further was investigated. Trials which imitated natural contamination were designed for and evaluated in an experimental process-line within an enterprise. The applications by immersion were composed of hot water, hot lactic acid, hot and cool water and chlorinated water. Concerning the industrial tests, vacuum tumbling and freezing were further process steps. In terms of reduction of the microbial counts, immersion in a hot lactic acid solution did enhance the antimicrobial effect. The application of a 2-3% hot lactic acid immersion at 70 and 75◦C for 10 - 30 seconds contact time was able to reduce the number of Salmonella on artificially contaminated chicken breast surfaces significantly more effectively compared to water immersion. The highest reduction of Salmonella (2.01 log10 MPN per gram meat) was achieved by a 3% lactic acid treatment at 75◦C for 20 seconds contact time. Hot water treatment alone eliminated 1 – 2 log10 CFU Salmonella Typhimurium per ml pure culture and 1 log10 CFU per gram contaminated meat. Notably, bacterial reductions achieved in the experiments in the enterprise were lower than in the laboratory experiments. The ranges of bacterial reductions by heat treatment at 80◦C for 15 - 30 seconds with and without 3% lactic acid solution were for mesophilic aerobic bacteria between 0.2 to 0.3 and 0.1 to 0.3, for Enterobacteriaceae between 0.1 to 0.8 and 0.1 to 0.4, for coliforms between 0.4 to 0.9 and 0.0 to 0.4, and for pseudomonads between 0.4 to 0.6 and 0.0 to 0.4 log10 CFU per gram meat for each of the two respective treatments. Vacuum tumbling employed before the immersion in 3% lactic acid soluion (TLHL) did effectively decontaminate the natural contamination with mesophilic aerobic bacteria and with pseudomonads (p≤ 0.05). Similarly, vacuum tumbling used after the hot water immersion (HLT) also significantly reduced natural contaminations, but for Enterobacteriaceae and coliforms in this case. Hot water followed by cool water in the enterprise experiments resulted in a reduction of bacterial counts of mesophilic aerobic bacteria of 0.02 to 0.53, of Enterobacteriaceae of 0.04 to 0.28, of coliforms of 0.10 to 0.37 and of pseudomonads of 0.08 to 0.96 log10CFU per gram chicken meat. Two-time treatment with hot water at 70°C for 15 seconds and cool water, followed by tumbling and freezing, did reduce bacteria more effectively than one-time treatment with hot water followed by cool water. Based on these low antimicrobial effects, the following immersion methods are not recommended for practical application: hot water at 80°C for 15 seconds (HW1); hot water at 75°C for 15 seconds and then cool water followed by tumbling for 15 minutes (HCS2 and HCK2); two-times treatment with hot water at 70°C, 15 seconds and cool water (HCD2) without tumbling. In terms of the microstructure of meat, collagen fibers started to denature at 80°C for 15 seconds in a hot lactic acid immersion and at 80°C for 20 seconds in a hot water immersion without lactic acid. With regard to the sensory attributes of treated chicken breast surfaces, aiming at results that almost equal the visual surface of raw chicken meat, the best treatment was two-time hot and cool water immersion. In contrast, TLHL clearly did reduce the natural bacterial contamination of chicken breast. Unfortunately, this method caused unacceptable taste and appearance changes. Treatment with 3% lactic acid did not make the meat sour but led to a change of the meat texture. In the chemical treatment experiment, chlorinated water inactivated Salmonella Typhimurium in culture 3 log10 CFU per ml. However, chlorinated water did not reduce bacteria on chicken breasts very efficiently. Additionally adverse sensory effects occurred. Concerning the decontamination of raw chicken breasts minimizing a compromise must be found for minimizing tissue damage and sensory alterations on the one hand and maximizing antimicrobial effects on the other hand.Labor- und Praxisversuche zur Reduktion der Salmonellenkontamination von rohen Hühnerbrustfilets Die Arbeit diente dem Ziel, ein geeignetes Dekontaminationsverfahren für Geflügelfleisch zu entwickeln. Hierfür wurden Laboruntersuchungen mit physikalischen und chemischen Methoden sowie deren Kombination durchgeführt. Die so gewonnenen Ergebnisse wurden weiterhin mit Hilfe dreier Versuchsreihen in einem Geflügelfleisch verarbeitenden Betrieb überprüft. Neben Erhebungen an Salmonella Typhimurium DSM 5569-Reinkulturen wurde bei den Laborstudien rohe Pectoralis-Muskulatur künstlich kontaminiert und in Test-Flüssigkeiten eingetaucht, wobei die Art des Mediums (Leitungswasser, Lösungen verschiedener organischer Säuren, chloriertes Wasser), die Behandlungsdauer und die Temperatur variierten. Bei den Feldversuchen kamen das Vakuum-Tumbeln und Einfrieren als mögliche Einflussfaktoren hinzu. Für die einzelnen Ansätze wurden außer der Keimzahlreduktion die sensorisch erfassbaren Merkmale wie Aussehen, Konsistenz, Geruch und Geschmack und z. T. mit Hilfe der histologischen Analyse auch der Verleimungsgrad des Kollagens ermittelt. Gemessen an den mit der kulturellen Technik nachweisbaren Keimzahlen zeigte im Modellversuch das Eintauchen in heiße Milchsäurelösung eine bessere Inaktivierung als der reine Abwascheffekt mit kaltem Leitungswasser. Bei den überprüften Varianten handelte es sich um 70 und 75°C heiße, 2 und 3 %ige Laktatlösung sowie Einwirkungszeiten von 10 – 30 Sekunden. Mit einer Verminderung der Salmonellendichte um 2.01 log10 MPN/g erbrachte die Kombination 3%ige Milchsäure/75°C/20 sec die stärkste Keimreduktion. Alleinige Anwendung von 70 bzw. 75°C heißem Wasser eliminierte 1-2 log10 KbE Salmonella Typhimurium/ml Reinkultur und 1 log10 KbE/g kontaminiertes Geflügelfleisch. Bei den Erhebungen im Verarbeitungsbetrieb fiel der Dekontaminationseffekt vergleichbarer Vorgehensweisen geringer als im Laborexperiment aus. Die Spannweiten der Keimzahl-reduktion beliefen sich bei der Prozessparameter- Kombination „80°C/15-30 sec/ ohne und mit 3 %ige Laktatlösung“ auf 0,1-0,3 bzw. 0,2-0,3 aerobe mesophile Gesamtkeime, 0,0-0,4 bzw. 0,4-0,6 Pseudomonaden, 0,1-0,4 bzw. 0,1-0,6 Enterobakteriazeen, 0,0-0,4 bzw. 0,4-0,9 für Coliforme, wobei die letzten beiden Gruppen als Indikatoren für Salmonellen dienten. Außerdem verminderte Vakuum-Tumbeln vor der Milchsäurebehandlung (TLHL) die Gesamtkeimzahl und Pseudomonadendichte signifikant (p<0.05), während Vakuum- Tumbeln nach der Immersion in Heißwasser (HLT) statistisch nachweisbar die Enterobakteriazeen- und Coliformenzahl senkte. Eine weiterhin im Betrieb überprüfte zweistufige Modifikation, bei der eine Kaltwasser- der 15 sec dauernden 70°C-Heißwassereinwirkung folgte, ergab eine Reduktion der Gesamtkeimzahl von 0,02-0,53, der Enterobakteriazeen von 0,04-0,28, der Coliformen von 0,10-0,37 und der Pseudomonaden von 0,08-0,96 KbE/g Brustfilet. Der inaktivierende Effekt ließ sich verstärken, indem diese Behandlung wiederholt wurde, gefolgt von den Prozessstufen Tumbeln und Einfrieren. Auf Grund ihrer geringen antimikrobiellen Wirkung empfehlen sich folgende der getesteten Varianten der Immersionstechnik nicht für die Praxis: 80°C heißes Wasser/15 sec (HW1), 75°C heißes Wasser/15 sec mit nachfolgender Kaltwasserbehandlung und 15-minütigem Tumbeln (HCS2 und HCK2), zweimaliges Einwirkeng von 70°C heißem Wasser/15 sec mit anschließender Kühlung ohne Tumbeln (HCD2). Im Hinblick auf die Mikrostruktur des Fleisches begann die Denaturierung und Quellung der kollagenen Fasern bei 80°C Milchsäurelösung/15 sec bzw. 80°C Trinkwasser/20 sec. Zweimaliges Einwirken von 70°C heißem Wasser und Kaltwasserabkühlung bewirkte die geringsten optischen Veränderungen der rohen Fleischoberfläche. Die Kombination aus Vakuumtumbeln und nachfolgender 3%iger Laktatlösung zeigte allerdings eine deutlich bessere mikrobizide Wirkung, doch wurden Aussehen und Textur zu stark verändert. Zwar verringerte chloriertes Wasser die Salmonellendichte in Reinkulturen um 3 log10 KbE/ml, doch war dieser Effekt bei kontaminierter Geflügelbrust deutlich abgeschwächt. Darüberhinaus traten merkliche sensorische Abweichungen auf. Insgesamt muss bei jeglicher Dekontamination von rohem Geflügelfleisch ein Kompromiss zwischen der „erwünschten Denaturierung von mikrobiellem Eiweiß und der unerwünschten Denaturierung von Fleischeiweiß“ gefunden werden. Im Gegensatz zu vielen anderen Nationen wären die EG-Länder mit dieser Problematik jedoch erst konfrontiert, wenn die Gemeinschaft ihre kritische Haltung zur chemischen Dekontamination aufgeben würde. Angesichts weiterhin relevanter Salmonella- Prävalenzen bei Schlachtgeflügel scheint ein Umdenken nicht ausgeschlossen zu sein

Effect of Temperature on the Expression of Classical Enterotoxin Genes among Staphylococci Associated with Bovine Mastitis

Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p &lt; 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP

Decontamination of Pathogenic and Spoilage Bacteria on Pork and Chicken Meat by Liquid Plasma Immersion

In this research, we aimed to reduce the bacterial loads of Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, and Pseudomonas aeruginosa in pork and chicken meat with skin by applying cold plasma in a liquid state or liquid plasma. The results showed reductions in S. Enteritidis, S. Typhimurium, E. coli, and C. jejuni on the surface of pork and chicken meat after 15 min of liquid plasma treatment on days 0, 3, 7, and 10. However, the efficacy of the reduction in S. aureus was lower after day 3 of the experiment. Moreover, P. aeruginosa could not be inactivated under the same experimental conditions. The microbial decontamination with liquid plasma did not significantly reduce the microbial load, except for C. jejuni, compared with water immersion. When compared with a control group, the pH value and water activity of pork and chicken samples treated with liquid plasma were significantly different (p ≤ 0.05), with a downward trend that was similar to those of the control and water groups. Moreover, the redness (a*) and yellowness (b*) values (CIELAB) of the meat decreased. Although the liquid plasma group resulted in an increase in the lightness (L*) values of the pork samples, these values did not significantly change in the chicken samples. This study demonstrated the efficacy of liquid plasma at reducing S. Enteritidis, S. Typhimurium, E. coli, C. jejuni, and S. aureus on the surface of pork and chicken meat during three days of storage at 4–6 °C with minimal undesirable meat characteristics

Qualitative risk assessment of transmission pathways of highly pathogenic avian influenza (HPAI) virus at live poultry markets in Dhaka city, Bangladesh

Analysis of environmental samples obtained from the Live Poultry Markets (LPMs) of Dhaka City, Bangladesh, has revealed that the highest degree of prevalence of highly pathogenic avian influenza A (HPAI, H5N1), besides other subtypes of the LPAI virus, poses the plausible risk of transmission of these viruses between human and poultry species. The present study was conducted using the OIE risk analysis framework to assess the risk level of each pathway successively. The estimated risk parameters were integrated towards to obtain the overall risk level for each specific HPAI transmission pathway using the matrix adapted by Cristobel Zepeda accompanying other expert consultations. The relevant data obtained from published and unpublished sources, together with survey data of field observations, were used to formulate and confirm the risk pathways and their associated risks. The results revealed that the risk of the release of the HPAI virus was medium when exposure was high. Additionally, the consequence would be considered very high with a medium degree of uncertainty for all parameters. Ultimately, the overall risk for transmission was estimated as medium with a medium degree of uncertainty. The findings of this study reveal that there is a significant threat that HPAI virus transmission could occur among poultry and humans and effectively sustain within the environment of the LPMs. Our findings are primarily focused on public health considerations, the hygienic slaughter of poultry and the relevant cleaning and sanitation practices conducted in the LPMs to support evidence-based decision-making processes. The findings of the study have the potential to be used to formulate effective risk reduction measures and can be further adapted in low-resource settings without major infrastructural changes required of the LPMs. All of which would reduce the risk of HPAI virus release and further lessen the degree of exposure and transmission in established LPMs