Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Impact of low-dose ozone nanobubble treatments on antimicrobial resistance genes in pond water.

Antimicrobial resistance (AMR) poses a significant global health threat as the silent pandemic. Because of the use of antimicrobials in aquaculture systems, fish farms may be potential reservoirs for the dissemination of antimicrobial resistance genes (ARGs). Treatments with disinfectants have been promoted to reduce the use of antibiotics; however, the effect of these types of treatments on AMR or ARGs is not well known. This study aimed to evaluate the effects of low dose ozone treatments (0.15 mg/L) on ARG dynamics in pond water using metagenomic shotgun sequencing analysis. The results suggested that ozone disinfection can increase the relative abundance of acquired ARGs and intrinsic efflux mediated ARGs found in the resistance nodulation cell division (RND) family. Notably, a co-occurrence of efflux and non-efflux ARGs within the same bacterial genera was also observed, with most of these genera dominating the bacterial population following ozone treatments. These findings suggest that ozone treatments may selectively favor the survival of bacterial genera harboring efflux ARGs, which may also have non-efflux ARGs. This study underscores the importance of considering the potential impacts of disinfection practices on AMR gene dissemination particularly in aquaculture settings where disinfectants are frequently used at low levels. Future endeavors should prioritize the evaluation of these strategies, as they may be associated with an increased risk of AMR in aquatic environments.

Epidemiology of Salmonella enterica subspecies enterica serotypes, isolated from imported, farmed and feral poultry in the Cayman Islands.

Non-typhoidal Salmonellae (NTS) are common foodborne pathogens throughout the world causing acute gastroenteritis. Compared to North America and Europe, there is little information on NTS in the Caribbean. Here we investigated the prevalence and characteristics of NTS present in the local poultry of the Cayman Islands to determine the public health risk. In total, we collected 156 samples. These were made up of boot swabs of 31 broiler farms and 31 layer farms (62 samples), paper bedding from 45 imported chick boxes, and 49 pooled cecum samples from feral chickens, each sample representing 10 individual chickens. Salmonella was isolated using the ISO 6579 protocol and isolates were characterized using Whole Genome Sequencing (WGS) analysis. Eighteen Salmonella isolates were obtained and comprised six S. enterica subspecies enterica serotypes and one subspecies houtenae serotype. Serotypes were: S. Kentucky (n = 9), S. Saintpaul (n = 5), S. Javiana (n = 1), S. Senftenberg (n = 1), S. Poona (n = 1) and S. Agona (n = 1). S. Kentucky strains were all ST152 and clonally related to poultry strains from the United states. S. Saintpaul ST50 strains showed clonality to North American strains. Over half of the strains (n = 11) contained resistance genes to at least two antibiotic groups and five strains were MDR, mainly those from imported day-old chicks. The blaCMY-2 gene was found in S. Kentucky from day-old chicks. Strains from feral poultry had no acquired AMR genes. While serotypes from feral poultry have been identified in human infections, they pose minimal risk due to their low virulence.

Prevalence and characterization of antimicrobial-resistant Escherichia coli in chicken meat from wet markets in Hong Kong.

Given the close contact between animals, animal products, and consumers in wet markets, fresh meat products are considered a potential source and disseminator of antimicrobial-resistant (AMR) bacteria near the end of the food chain. This cross-sectional study was conducted to estimate the prevalence of select AMR-E. coli in fresh chicken meat collected from wet markets in Hong Kong and to determine target genes associated with the observed resistance phenotypes. Following a stratified random sampling design, 180 fresh half-chickens were purchased from 29 wet markets across Hong Kong in 2022 and immediately processed. After incubation, selective isolation was performed for extended-spectrum ß-lactamase producing (ESBL), carbapenem-resistant (CRE), and colistin-resistant (CSR) E. coli. The bacterial isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Disc Diffusion was used to determine the susceptibility of ESBL- and CRE-E. coli isolates. The broth microdilution method was used to determine the minimum inhibitory concentration of CSR-E. coli. Targeted resistance genes were then detected by PCR. The prevalence of ESBL-E. coli and CSR-E. coli were estimated at 88.8% (95% CI: 83.4-93.1%) and 6.7% (95% CI: 3.5-11.4%), respectively. No CRE-E. coli isolate was detected. The blaCTX-M-1 gene was the most common ß-lactamase group in isolated E. coli (80%), followed by blaTEM (63.7%); no blaSHV gene was detected. Forty-five percent of the isolates had blaTEM and blaCTX-M-1 simultaneously. The mcr-1 gene was detected in all 12 CSR isolates. Of 180 meat samples, 59 were from Mainland China, and 121 were locally sourced. There was no statistically significant difference in the prevalence of ESBL- and CSR-E. coli between the two sources. Our findings can be used to inform food safety risk assessments and set the stage for adopting targeted control and mitigation measures tailored to the local wet markets.

Draft genomes of 16 Bartonella henselae strains from cats in Valdivia, Chile.

Bartonella henselae is a primary zoonotic agent, having cats as asymptomatic reservoirs. In humans, it causes cat scratch disease. Here, we report the whole genome sequences of 16 strains isolated from cats in Valdivia city, Southern Chile. Strains showed little variability in the multilocus sequence typing profiles.

The Diversity and Zoonotic Potential of Staphylococcus pseudintermedius in Humans and Pet Dogs in Trinidad and Tobago.

Staphylococcus pseudintermedius is an opportunistic pathogen that is frequently isolated from canines. It is of escalating interest because of its increasing antimicrobial resistance and zoonotic potential. Although many published articles are available that describe isolates obtained from diseased dogs and humans, this study focused on isolates obtained from healthy dogs and their owners who presented at clinics for routine veterinary care and utilized whole genome sequencing-based analyses for strain comparisons. A total of 25 humans and 27 canines were sampled at multiple sites, yielding 47 and 45 isolates, respectively. Whole genome sequence analysis was performed. We detected mostly new sequence types (STs) and a high diversity. Strains carried few antimicrobial resistance genes and plasmids, albeit three MRSP strains were found that belonged to two internationally distributed STs. The virulence content did not provide insights toward a tendency to colonization of humans but supported that there may be differences in the surface proteins between carrier strains and those causing pyoderma. We identified 13 cases in which humans were infected with strains from the dog they owned.

Antimicrobial Use and Resistance in Animals from a One Health Perspective.

Among the many global health issues, antimicrobial resistance (AMR) is one that exemplifies the One Health approach, defined as a joint effort in which multiple disciplines collaborate to provide solutions for human, animal, and environmental health [...].

Virulence and Antimicrobial Resistance Profiling of Salmonella Serovars Recovered from Retail Poultry Offal in KwaZulu-Natal Province, South Africa.

As poultry organ meat is widely consumed, especially in low- and middle-income countries, there is reason to investigate it as a source of Salmonella infections in humans. Consequently, the aim of this study was to determine the prevalence, serotypes, virulence factors and antimicrobial resistance of Salmonella isolated from chicken offal from retail outlets in KwaZulu-Natal, South Africa. Samples (n = 446) were cultured for the detection of Salmonella using ISO 6579-1:2017. Presumptive Salmonella were confirmed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Salmonella isolates were serotyped using the Kauffmann-White-Le Minor scheme and antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion technique. A conventional PCR was used for the detection of Salmonella invA, agfA, lpfA and sivH virulence genes. Of the 446 offal samples, 13 tested positive for Salmonella (2.91%; CI = 1.6-5). The serovars included S. Enteritidis (n = 3/13), S. Mbandaka (n = 1/13), S. Infantis (n = 3/13), S. Heidelberg (n = 5/13) and S. Typhimurium (n = 1/13). Antimicrobial resistance against amoxicillin, kanamycin, chloramphenicol and oxytetracycline was found only in S. Typhimurium and S. Mbandaka. All 13 Salmonella isolates harboured invA, agfA, lpfA and sivH virulence genes. The results show low Salmonella prevalence from chicken offal. However, most serovars are known zoonotic pathogens, and multi-drug resistance was observed in some isolates. Consequently, chicken offal products need to be treated with caution to avoid zoonotic Salmonella infections.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococci (MRS) and Mammaliicocci (MRM) in Dromedary Camels from Algeria: First Detection of SCCmec-mecC Hybrid in Methicillin-Resistant Mammaliicoccus lentus.

Dromedary camels are an important source of food and income in many countries. However, it has been largely overlooked that they can also transmit antibiotic-resistant bacteria. The aim of this study was to identify the Staphylococcaceae bacteria composition of the nasal flora in dromedary camels and evaluate the presence of methicillin-resistant Mammaliicoccus (MRM) and methicillin-resistant Staphylococcus (MRS) in dromedary camels in Algeria. Nasal swabs were collected from 46 camels from seven farms located in two different regions of Algeria (M'sila and Ouargla). We used non-selective media to determine the nasal flora, and antibiotic-supplemented media to isolate MRS and MRM. The staphylococcal isolates were identified using an Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS). The mecA and mecC genes were detected by PCR. Methicillin-resistant strains were further analysed by long-read whole genome sequencing (WGS). Thirteen known Staphylococcus and Mammaliicoccus species were identified in the nasal flora, of which half (49.2%) were coagulase-positive staphylococci. The results showed that four out of seven farms were positive for MRS and/or MRM, with a total of 16 isolates from 13 dromedary camels. The predominant species were M. lentus, S. epidermidis, and S. aureus. Three methicillin-resistant S. aureus (MRSA) were found to be ST6 and spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 was the predominant ST identified. Phylogenetic analysis showed clonal relatedness among M. lentus strains, while S. epidermidis strains were not closely related. Resistance genes were detected, including mecA, mecC, ermB, tet(K), and blaZ. An SCCmec type VIII element was found in a methicillin-resistant S. hominis (MRSH) belonging to the ST1 strain. An SCCmec-mecC hybrid element was detected in M. lentus, similar to what was previously detected in M. sciuri. This study highlights that dromedary camels may be a reservoir for MRS and MRM, and that they contain a specific set of SCCmec elements. This emphasizes the need for further research in this ecological niche from a One Health perspective.

The Endometrial Microbiota-16S rRNA Gene Sequence Signatures in Healthy, Pregnant and Endometritis Dairy Cows.

Endometritis is one of the most important causes of infertility in dairy cows, resulting in high economic losses in the dairy industry. Though the presence of a commensal uterine microbiota is now well established, the complex role of these bacteria in genital health, fertility, and susceptibility to uterine diseases remains unclear. In this study, we explore the endometrial microbiota through 16S rRNA gene profiling from cytobrush samples taken ex vivo from healthy, pregnant, and endometritis cows. There were no significant differences between healthy and pregnant cows, whose uterine microbiota were dominated by Streptococcus, Pseudomonas, Fusobacterium, Lactococcus and Bacteroides. Compared to pregnant and clinically healthy cows, the uterine bacterial community of endometritis cows was significantly decreased in species diversity (p < 0.05), reflecting uneven community composition in different patterns with either dominance of Escherichia-Shigella, Histophilus, Bacteroides and Porphyromonas or Actinobacteria.

Current Insights Regarding the Role of Farm Animals in the Spread of Antimicrobial Resistance from a One Health Perspective.

Antimicrobial resistance (AMR) represents a global threat to both human and animal health and has received increasing attention over the years from different stakeholders. Certain AMR bacteria circulate between humans, animals, and the environment, while AMR genes can be found in all ecosystems. The aim of the present review was to provide an overview of antimicrobial use in food-producing animals and to document the current status of the role of farm animals in the spread of AMR to humans. The available body of scientific evidence supported the notion that restricted use of antimicrobials in farm animals was effective in reducing AMR in livestock and, in some cases, in humans. However, most recent studies have reported that livestock have little contribution to the acquisition of AMR bacteria and/or AMR genes by humans. Overall, strategies applied on farms that target the reduction of all antimicrobials are recommended, as these are apparently associated with notable reduction in AMR (avoiding co-resistance between antimicrobials). The interconnection between human and animal health as well as the environment requires the acceleration of the implementation of the 'One Health' approach to effectively fight AMR while preserving the effectiveness of antimicrobials.

Presence of Methicillin Resistant Staphylococcus aureus (MRSA) in Feces of the Small Indian Mongooses (Urva auropunctata) on Saint Kitts and Nevis, West Indies.

Although, historically, Methicillin-Resistant Staphylococcus aureus (MRSA) was restricted to humans, since 2005 these strains emerged in livestock and wildlife. Therefore, a One Health approach was applied to analyze the diversity and characteristics of S. aureus strains isolated from the invasive species of mongoose (Urva auropunctata) in St. Kitts. Fecal samples collected from these animals (n = 81) were cultured on selective agar. The isolated S. aureus strains were identified using MALDI-TOF and further characterized by whole genome sequence analysis. The fecal microbiome study identified the presence of S. aureus in 5 animals. Both MSSA (n = 3) and MRSA (n = 2) strains were identified. The two MRSA isolated were nearly identical ST5 SCCmec IVa (2B) strains. The two MSSA isolated were a new ST7434, pertaining to clonal complex 30, and the other belonged to ST5, but unrelated to the MRSA ST5. The SCCmec IVa (2B) is, however, the main SCCmec in human MRSA of different STs identified in St Kitts, indicating potential horizontal transmission events. In conclusion, a new type of MSSA, ST7434, was found and MRSA ST5 t002 SCCmec IVa (2B) found its way into wildlife on a small Caribbean Island. Further One Health studies are necessary to determine the role of MRSA in wildlife.

Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Methicillin-Resistant Staphylococci and Mammaliicoccus (MRNaS) Associated with Animals and Food Products in Arab Countries: A Review.

The prevalence of methicillin resistance in staphylococci has been increasing globally and is currently one of the major public health concerns. In particular, treating infections caused by staphylococci with acquired antimicrobial resistance is problematic, as their treatment is more difficult. The resistance is found both in human and animal staphylococcal strains. Methicillin-resistant staphylococci (MRS) have also been increasingly reported in wildlife. In Arab countries, MRS has been detected in food producing animals and food products; however, the risk this poses is somewhat unclear, and still a significant lack of information on the trend and distribution of these pathogens in these countries, which have a specific ecosystem (desert) and traditions (Muslim culture). In this manuscript, we aim to provide an overview of the prevalence and the major MRS clonal lineages circulating in these specific countries and compare to them other situations with different ecosystems and cultures.

Antimicrobial Resistance, Enterotoxin and mec Gene Profiles of Staphylococcus aureus Associated with Beef-Based Protein Sources from KwaZulu-Natal Province, South Africa.

Annually, approximately 23,000 cases of food poisoning by Staphylococcus aureus enterotoxins are reported worldwide. The aim of this study was to determine the occurrence and characterize S. aureus on beef and beef products in South Africa. Organ meats (n = 169), raw processed meat (n = 110), raw intact (n = 53), and ready-to-eat meats (n = 68) were obtained from 25 retail outlets. S. aureus was isolated and enumerated according to the ISO 6888-1 method. Identification of the strains was performed by MALDI-TOF MS. The antimicrobial resistance was determined using the disc diffusion test. The presence of methicillin-resistance genes and the staphylococcal enterotoxin genes was determined by PCR. Prevalence was low (13/400; CI 1.7-5) and all but one positive sample were from organ meats. Eight isolates were resistant to at least one antibiotic. Two isolates carried the mecC gene. All the isolates tested positive for seg, seh, sei, and sep, whilst 53.8% were positive for sea. None of the isolates was positive for ser, sej, seb, sec, or sed. The prevalence of S. aureus was low, with organ meats being the most contaminated. The presence of mecC-positive MRSA and of enterotoxins warrants further investigation and risk assessment.

Spontaneous Phage Resistance in Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide. The emergence of multidrug-resistant pathogens has renewed the interest in the therapeutic use of bacteriophages (phages). However, a major concern for the successful implementation of phage therapy is the emergence of phage-resistant mutants. The understanding of the phage-host interactions, as well as underlying mechanisms of resistance, have shown to be essential for the development of a successful phage therapy. Here, we demonstrate that the strictly lytic Escherichia phage vB_EcoM-P10 rapidly selected for resistance in the APEC ST95 O1 strain AM621. Whole-genome sequence analysis of 109 spontaneous phage-resistant mutant strains revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of these, a single SNP was detected while two SNPs were identified in a total of nine strains. In total, 34 unique SNPs were detected. In 42 strains, including 18 strains with SNP(s), gene losses spanning 17 different genes were detected. Affected by genetic changes were genes known to be involved in phage resistance (outer membrane protein A, lipopolysaccharide-, O- antigen-, or cell wall-related genes) as well as genes not previously linked to phage resistance, including two hypothetical genes. In several strains, we did not detect any genetic changes. Infecting phages were not able to overcome the phage resistance in host strains. However, interestingly the initial infection was shown to have a great fitness cost for several mutant strains, with up to ∼65% decrease in overall growth. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in APEC. Although acquired resistance to phages is frequently observed in pathogenic E. coli, it may be associated with loss of fitness, which could be exploited in phage therapy.

Classification of In Vitro Phage-Host Population Growth Dynamics.

The therapeutic use of bacteriophages (phage therapy) represents a promising alternative to antibiotics to control bacterial pathogens. However, the understanding of the phage-bacterium interactions and population dynamics seems essential for successful phage therapy implementation. Here, we investigated the effect of three factors: phage species (18 lytic E. coli-infecting phages); bacterial strain (10 APEC strains); and multiplicity of infection (MOI) (MOI 10, 1, and 0.1) on the bacterial growth dynamics. All factors had a significant effect, but the phage appeared to be the most important. The results showed seven distinct growth patterns. The first pattern corresponded to the normal bacterial growth pattern in the absence of a phage. The second pattern was complete bacterial killing. The remaining patterns were in-between, characterised by delayed growth and/or variable killing of the bacterial cells. In conclusion, this study demonstrates that the phage-host dynamics is an important factor in the capacity of a phage to eliminate bacteria. The classified patterns show that this is an essential factor to consider when developing a phage therapy. This methodology can be used to rapidly screen for novel phage candidates for phage therapy. Accordingly, the most promising candidates were phages found in Group 2, characterised by growth dynamics with high bacterial killing.

Salmonella in Pig Farms and on Pig Meat in Suriname.

Salmonella is one of the most important food borne zoonotic pathogens. While mainly associated with poultry, it has also been associated with pigs. Compared to the high-income countries, there is much less known on the prevalence of Salmonella in low- and middle-income countries, especially in the Caribbean area. Therefore, we investigated the prevalence of Salmonella in pigs and pig meat in Suriname. A total of 53 farms and 53 meat samples were included, and Salmonella was isolated using standard protocols. Strains were subjected to whole genome sequencing. No Salmonella was found on pig meat. Five farms were found to be positive for Salmonella, and a total of eight different strains were obtained. Serotypes were S. Anatum (n = 1), S. Ohio (n = 2), a monophasic variant of S. Typhimurium (n = 3), one S. Brandenburg, and one S. Javaniana. The monophasic variant of S. Typhimurium belonged to the ST34 pandemic clone, and the three strains were very similar. A few resistance genes, located on mobile genetic elements, were found. Several plasmids were detected, though only one was carrying resistance genes. This is the first study on the prevalence of Salmonella in pigs in the Caribbean and that used whole genome sequencing for characterization. The strains were rather susceptible. Local comparison of similar serotypes showed a mainly clonal spread of certain serotypes.

Concurrent Resistance to Carbapenem and Colistin Among Enterobacteriaceae Recovered From Human and Animal Sources in Nigeria Is Associated With Multiple Genetic Mechanisms.

Resistance to last resort drugs such as carbapenem and colistin is a serious global health threat. This study investigated carbapenem and colistin resistance in 583 non-duplicate Enterobacteriaceae isolates utilizing phenotypic methods and whole genome sequencing (WGS). Of the 583 isolates recovered from humans, animals and the environment in Nigeria, 18.9% (110/583) were resistant to at least one carbapenem (meropenem, ertapenem, and imipenem) and 9.1% (53/583) exhibited concurrent carbapenem-colistin resistance. The minimum inhibitory concentrations of carbapenem and colistin were 2-32 µg/mL and 8 to >64 µg/mL, respectively. No carbapenem resistant isolates produced carbapenemase nor harbored any known carbapenemase producing genes. WGS supported that concurrent carbapenem-colistin resistance was mediated by novel and previously described alterations in chromosomal efflux regulatory genes, particularly mgrB (M1V) ompC (M1_V24del) ompK37 (I70M, I128M) ramR (M1V), and marR (M1V). In addition, alterations/mutations were detected in the etpA, arnT, ccrB, pmrB in colistin resistant bacteria and ompK36 in carbapenem resistant bacteria. The bacterial isolates were distributed into 37 sequence types and characterized by the presence of internationally recognized high-risk clones. The results indicate that humans and animals in Nigeria may serve as reservoirs and vehicles for the global spread of the isolates. Further studies on antimicrobial resistance in African countries are warranted.

Prevalence and Characteristics of Staphylococcus aureus Associated with Meat and Meat Products in African Countries: A Review.

Antimicrobial resistance has been increasing globally, which negatively affects food safety, veterinary, and human medicine. Ineffective antibiotics may cause treatment failure, which results in prolonged hospitalisation, increased mortality, and consequently, increased health care costs. Staphylococcus aureus causes a diverse range of infections including septicaemia and endocarditis. However, in food, it mainly causes food poisoning by the production of enterotoxins. With the discovery of methicillin-resistant S. aureus strains that have a separate reservoir in livestock animals, which were termed as livestock-associated methicillin-resistant S. aureus (LA-MRSA) in 2005, it became clear that animals may pose another health risk. Though LA-MRSA is mainly transferred by direct contact, food transmission cannot be excluded. While the current strains are not very pathogenic, mitigation is advisable, as they may acquire new virulence genes, becoming more pathogenic, and may transfer their resistance genes. Control of LA-MRSA poses significant problems, and only Norway has an active mitigation strategy. There is limited information about LA-MRSA, MRSA in general, and other S. aureus infections from African countries. In this review, we discuss the prevalence and characteristics of antimicrobial susceptible and resistant S. aureus (with a focus on MRSA) from meat and meat products in African countries and compare it to the situation in the rest of the world.