Search Results (91)

Poultry feces, a critical biomarker for health assessment, requires timely and accurate pathological identification for food safety. Conventional visual-only methods face limitations due to environmental sensitivity and high visual similarity among feces from different diseases. To address this, we propose MMCD (Multimodal Chicken-feces Diagnosis), a ResNet50-based multimodal fusion model leveraging semantic complementarity between images and descriptive text to enhance diagnostic precision. Key innovations include the following: (1) Integrating MASA(Manhattan self-attention)and DSconv (Depthwise Separable convolution) into the backbone network to mitigate feature confusion. (2) Utilizing a pre-trained BERT to extract textual semantic features, reducing annotation dependency and cost. (3) Designing a lightweight Gated Cross-Attention (GCA) module for dynamic multimodal fusion, achieving a 41% parameter reduction versus cross-modal transformers. Experiments demonstrate that MMCD significantly outperforms single-modal baselines in Accuracy (+8.69%), Recall (+8.72%), Precision (+8.67%), and F1 score (+8.72%). It surpasses simple feature concatenation by 2.51–2.82% and reduces parameters by 7.5M and computations by 1.62 GFLOPs versus the base ResNet50. This work validates multimodal fusion’s efficacy in pathological fecal detection, providing a theoretical and technical foundation for agricultural health monitoring systems. Full article

Chicken anemia virus (CAV) and Eimeria tenella (E. tenella) are economically important pathogens of the poultry industry worldwide. However, the impact of dual infection of these two pathogens in chickens remains unclear. This study investigated the pathogenic effects of dual infection with CAV and E. tenella using two trials. In Trial A, chickens were infected at 21 days of age (D21) with either CAV and E. tenella simultaneously (C₂₁ + T₂₁), CAV alone (C_21a), E. tenella alone (T₂₁), or PBS as a negative control (NC). In Trial B, chickens received CAV at D21 followed by E. tenella at D28 (C₂₁ + T₂₈), CAV alone at D21 (C_21b), E. tenella alone at D28 (T₂₈), or PBS at D21 (NC). Assays of lesion scores (LS), oocysts per gram (OPG) of feces, packed cell volume (PCV), and thymus index (TI) were used to assess variations in pathogenicity. Both the C₂₁ + T₂₁ and C₂₁ + T₂₈ groups showed higher OPG than the group infected with E. tenella alone, with significantly elevated OPG in the secondary infection scenario and more severe lesions in the concurrent co-infection group (p < 0.05). Anemia, indicated by PCV < 27%, was observed in the C₂₁ + T₂₁ group at day 28 and in the C₂₁ + T₂₈ group at day 35, both of which had significantly lower PCV values than the group infected with CAV alone (p < 0.001). Thymus atrophy was most severe in C₂₁ + T₂₁ at 28 days old (p < 0.05; p < 0.01). In this study, preliminary observations suggested that concurrent and secondary infections with CAV and E. tenella showed variable trends that may indicate potential interactions; however, these exploratory findings require more systematic validation in older chickens. Full article

This study aimed to isolate and genetically characterize Arcobacter species from broiler chickens sampled at three slaughterhouses in Grenada, West Indies. A total of 126 samples—including cloacal swabs, intestinal contents, and meat—from 42 birds were cultured using a chromogenic agar medium. Arcobacter spp. were detected in 21.4% (9/42) of the birds. Among the sample types, meat exhibited the highest prevalence at 14.3% (6/42), followed by fecal samples at 7.1% (3/42) and cloacal swabs at 2.4% (1/42). Genus- and species-specific polymerase chain reaction assays on 33 isolates identified five Arcobacter species: A. butzleri, A. cryaerophilus, and A. skirrowii (each 18.2%), as well as A. cibarius and A. thereius (each 6.1%). Genetic diversity was further assessed via Enterobacterial Repetitive Intergenic Consensus–polymerase chain reaction, which revealed 13 distinct genotypic fingerprints forming six clusters, with a high discriminatory power (D = 0.96). This study represents the first documented isolation and molecular characterization of five Arcobacter species from broiler chickens in Grenada across multiple sample types. These findings underscore the zoonotic implications of isolating Arcobacter spp., particularly in contaminated poultry meat destined for human consumption. The presence of Arcobacter spp. in poultry carcasses poses a significant public health concern. To mitigate this public health risk, recommendations include surveillance for the presence of this pathogen in Hazard Analysis and Critical Control Points plans or other tools used to identify pathogens compromising food safety and public health. Full article

Newcastle disease viruses (NDVs) circulating among wild birds and poultry may differ in virulence. Some NDVs cause devastating outbreaks in chickens. The NDV/duck/Moscow/3639/2008 (d3639) strain was isolated from a wild duck. Its genome was sequenced (PP795281, GenBank) and the biological properties, specifically for infection in chicken and mice, were studied. Strain d3639 of genotype I.2 has an F protein cleavage site (112-GKQGRL-117) and a HN protein length (616 a.a.) of the lentogenic pathotype. It was tested, in comparison with the genotype II LaSota vaccine strain, for its immunogenicity and protective efficacy against a challenge with the velogenic NDV strain NDV/chicken/Moscow/6081/2022 (ch6081) of sub-genotype VII.1.1, the complete genome of which was also sequenced in this study (PP766718, GenBank). Both the d3639 and LaSota viruses did not induce clinical signs in chickens or mice. Single immunization was performed by inoculation through drinking water with the live virus. Inoculation protected the chickens during a subsequent challenge with velogenic ch6081 and significantly reduced shedding in feces. Double immunization was sufficient to achieve prolonged immunity and prevented the shedding of the velogenic virus after the challenge. Thus, this natural lentogenic d3639 virus possesses properties similar to the LaSota vaccine strain and can protect against sub-genotype VII.1.1 NDV. Full article

Background: The emergence of linezolid resistance, mediated by genes such as optrA and cfr(D), poses a growing public health threat. While these genes have been detected in clinical and animal-derived Enterococcus species, their presence in underexplored species like Enterococcus saccharolyticus remains undocumented, leaving a significant gap in our understanding of their dissemination and stability. Method: E. saccharolyticus GXN23C125Es was screened for the presence of known linezolid resistance genes via PCR analysis. Conjugation and stability experiments were used to evaluate the transferability and stability of the resistance genes. The complete genome of GXN23C125Es was obtained using both the Illumina and Nanopore platforms. Results: We report the first identification of optrA and cfr(D) in GXN23C125Es from chicken feces in China. Whole-genome sequencing revealed multiple plasmid-borne resistance genes, including optrA, cfr(D), fexA, and erm(A). Stability testing demonstrated that optrA was highly stable, while cfr(D) was rapidly lost without selective pressure. Conclusions: These findings highlight E. saccharolyticus as a potential reservoir for linezolid resistance genes, underscoring the need for enhanced surveillance of resistance determinants in animal-associated bacteria. Understanding the dissemination dynamics of optrA and cfr(D) is crucial for mitigating their impact on public health and guiding antimicrobial resistance management strategies. Full article

The disease caused by Salmonella pullorum has been demonstrated to exert a deleterious effect on the performance of poultry, giving rise to elevated mortality and considerable economic losses within the breeding industry. However, there is a paucity of research investigating the relationship between cecal gene expression and different isomer and Salmonella pullorum infection, and research on the relationship between intestinal microbiota and Salmonella pullorum infection is also limited. In this study, mRNA-Seq and metagenomic sequencing were performed on the cecal tissues and fresh feces of individuals who tested positive (n = 4) and negative (n = 4) for Salmonella pullorum, with the aim of exploring the chickens infected with Salmonella pullorum from two perspectives: the gene transcription level and the microbial level. The mRNA sequencing results revealed 1560 differentially expressed genes (DEGs), of which 380 genes were found to be up-regulated and 1180 genes were down-regulated. A number of genes were reported to be associated with immunity, including AQP8, SLC26A3, CBS, IFI6, DDX60, IL8L1 and IL8L2. Furthermore, a total of 1047 differentially expressed alternative splicings (DEASs) were identified through alternative splicing analysis, including CBS, SLC6A9, ILDR2, OCRL, etc. The joint analysis of DEGs and DEASs revealed 70 genes that exhibited both differentially expressed alternative splicings and differential expression, including CTNND1, TPM1, SPPL2A, etc. The results of metagenomic sequencing demonstrated that the abundances of Bacteroides, Firmicutes, and Verrucobacteria underwent a significant alteration subsequent to the infection of Salmonella pullorum. In summary, the present study conducted a preliminary exploration of the genetic basis of chickens infected with Salmonella pullorum. TPM1 and SPPL2A were found to be differentially expressed by mRNA-Seq, and differences in alternative splicing events. Furthermore, metagenomic sequencing revealed significant changes in the microbial communities of Bacteroidetes, Firmicutes, and Verrucobacteria during infection with Salmonella pullorum. Full article

The cytokines IFN-γ and CD154 have been well established, and they play pivotal roles in immune protection against Salmonella in mice, but their effects and specific mechanisms in Salmonella-infected chickens are less understood. In this study, we conducted animal experiments to screen the highly immunoprotective chIFN-γ-chCD154 fusion protein compared with single protein chIFN-γ or chCD154 in white Leghorn chickens. The results showed that compared with separate pretreatments with chIFN-γ and chCD154, the fusion protein, chIFN-γ-chCD154, synergistically increased survival of infected chickens, reduced bacterial load in feces and organs, and attenuated pathological damage to the liver and cecum. Pretreatment with chIFN-γ-chCD154 also increased humoral immune responses, expression of the tight junction proteins zo-1, occludin, and claudin-1, and the relative abundance of Enterococcus_cecorum, Lactobacillus_helveticus, and Lactobacillus_agilis, which protect against intestinal inflammation. Compared with single protein pretreatment, chIFN-γ-chCD154 significantly upregulated STAT1, IRF1, and GBP1 in infected chickens while decreasing mRNA expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-1β. In summary, damage to the cecal epithelial barrier and the inflammation induced by S. typhimurium infection was alleviated by chIFN-γ-chCD154 pretreatment through a mechanism involving the TLR4/MyD88/NF-κB and IFN-γ/STAT/IRF1/GBP1 pathways. Full article

Inappropriate antimicrobial use in food animal farming propels antimicrobial resistance (AMR) that affects all health domains. Colistin is a ‘Reserve’ antibiotic for human treatment to be conserved for multidrug-resistant pathogens; however, it is being used as an animal growth promoter in many developing countries. The evolution of mobilized colistin resistance (mcr) gene-mediated colistin resistance has been reported to be associated with rampant colistin use. This study investigated the current variants of the mcr gene in chicken gut contents in Bangladesh. A cross-sectional study was designed to assess the mcr-1 to mcr-5 genes in 80 fresh poultry droppings from commercial poultry farms and 40 poultry droppings from household farms. DNA was extracted from each poultry dropping using commercial kits (Qiagen GmbH, Hilden, Germany). Real-time quantitative polymerase chain reaction (RT-qPCR) was employed using the qTOWER3 thermal cycler (Analytik Jena GmbH, Jena, Germany) to analyze the mcr gene variants in the extracted DNA. This study observed that 47.5% (57/120) of the samples exhibited the presence of at least one mcr gene out of the five variants investigated. The individual detection rates of the mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were 42.5% (51/120), 2.5% (3/120), 1.7% (2/120), 5% (6/120), and 9.2% (11/120), respectively. The co-carriage of two or more genes was found in over 10% (10/57) of the samples. The triple occurrence of mcr genes was identified in three samples with the combination of mcr-1+mcr-2+mcr-4, mcr-1+mcr-3+mcr-5, and mcr-1+mcr-4+mcr-5. Overall, a significantly higher number of mcr genes were identified in the commercial farm chicken droppings compared to the household chicken droppings (p = 0.007). The existence of mcr genes in poultry feces in Bangladesh emphasizes the importance of proper poultry waste disposal and good hygiene practices in poultry livestock and its value chain. The potential impact of environmental ARGs should be considered in national and global policy documents. An integrated and combined approach to the One Health concept should be applied in all domains to understand and control the environment’s role in the evolution and transmission of AMR. Full article

This study investigates the effects of the Bacillus licheniformis (BL) ATCC 21424 strain, as a potential bacterial probiotic in broiler diets based on soybean meal (SBM) or cowpea seeds (CWP), on growth performance (GP), bone mineralization, and intestinal/fecal microbiota status (0 to 42 d age). A 2 × 2 factorial arrangement was employed in a completely randomized design, with four dietary treatments: SBM and CWP diets with or without BL supplementation (1.0 × 10¹¹ CFU spores g⁻¹ feed). A total of 480 one-day-old mixed-sex Ross 308 broiler chickens were randomly assigned to the treatments, with 6 pens of 20 chicks each. The results showed that broilers fed with CWP diets showed comparable body weight gain (BWG), feed intake (FI), and feed conversion rate to those fed the SBM diet (p > 0.05). The inclusion of BL improved BWG during the grower and finisher periods (p = 0.01) and overall study (p < 0.001), resulting in a numerical increase in FI (p = 0.054). In addition, BL in birds’ diets reduced abdominal fat (p = 0.032) and influenced cecum weight (p = 0.040). Additionally, BL improved tibia iron (Fe) and phosphorus (P) bone mineralization and reduced the calcium–phosphorus (Ca:P) ratio (p = 0.0001). Microbial analysis revealed that BL inclusion decreased Coliforms counts in the CWP diet (p = 0.073), reduced E. coli in the ileum (p ≤ 0.05), and lowered Clostridium spp. and Enterococcus spp. in the cecum broilers on SBM diets (p ≤ 0.05). The presence of Staphylococcus spp. in broiler feces was also reduced in both SBM and CWP groups (p < 0.05). In conclusion, the addition of BL to broiler diets enhanced growth performance and bone mineralization and positively influenced gut and excreta bacterial populations in both SBM and CWP diets. Full article

Herpesvirus of turkey (HVT) recombinant vector vaccines are widely used in the poultry industry. However, due to limitations in loading multiple foreign antigens into a single HVT vector, other viral vectors are urgently needed. Since chickens lack maternal immunity to duck enteritis virus (DEV), vector vaccines using DEV as a backbone are currently under study. Even though a recently developed DEV vector vaccine expressing the influenza hemagglutinin H5 of highly pathogenic avian influenza (DEV-H5) induces highly detectable anti-HA antibodies, safety issues hamper further vaccine development. In this work, tissue affinity and horizontal transmission in 1-day-old chickens were systematically evaluated after DEV-H5 vector vaccine inoculation. Sixty percent of DEV-H5-inoculated chickens died between day 2 and day 7 post-inoculation. The displayed clinical signs consisted of lethargy, anorexia, and diarrhea, and virus was shed in feces. Gross and/or histological lesions were recorded in the kidney, heart, intestine, liver, lung, and spleen. Moreover, DEV-H5 replication in intestinal cells caused an increment in interferon-α expression, while occluding junction proteins and ZO-1 expression were significantly upregulated. As a control, birds inoculated with a commercial recombinant turkey herpesvirus expressing the VP2 protein of the infectious bursal disease virus (HVT-VP2) vector vaccine showed neither clinical signs nor mortality. Overall, while the HVT-VP2 vaccine demonstrated complete safety in 1-day-old chickens, our potential DEV-H5 vaccine requires further attenuation for consideration as a vector vaccine candidate in chickens. Full article

Background/Objectives: The global emergence of antibiotic-resistant zooanthroponotic Escherichia coli strains, producing extended-spectrum beta-lactamases (ESBL-E) and persisting in the intestines of farm animals, has now led to the development of a pandemic of extra-intestinal infectious diseases in humans. The search for innovative probiotic microorganisms that eliminate ESBL-E from the intestines of humans and animals is relevant. Previously, we received three isolates of bifidobacteria: from milk of a calved cow (BLLT1), feces of a newborn calf (BLLT2) and feces of a three-year-old child who received fresh milk from this calved cow (BLLT3). Our goal was to evaluate the genetic identity of BLLT1, BLLT2, BLLT3 isolates using genomic DNA fingerprinting (GDF), to study the tolerance, adhesion, homeostatic and antibacterial activity of BLLT1 against ESBL-E. Methods: We used a complex of microbiological, molecular biological, and immunological methods, including next generation sequencing (NGS). Results: GDF showed that DNA fragments of BLLT2 and BLLT3 isolates were identical in number and size to DNA fragments of BLLT1. These data show for the first time the possibility of natural horizontal transmission of BLLT1 through with the milk of a calved cow into the intestines of a calf and the intestines of a child. BLLT1 was resistant to gastric and intestinal stresses and exhibited high adhesive activity to calf, pig, chicken, and human enterocytes. This indicates the unique ability of BLLT1 to inhabit the intestines of animals and humans. We are the first to show that BLLT1 has antibacterial activity against ESBL-E strains that persist in humans and animals. BLLT1 produced 145 ± 8 mM of acetic acid, which reduced the pH of the nutrient medium from 6.8 to 5.2. This had an antibacterial effect on ESBL-E. The genome of BLLT1 contains ABC-type carbohydrate transporter gene clusters responsible for the synthesis of acetic acid with its antibacterial activity against ESBL-E. BLLT1 inhibited TLR4 mRNA expression induced by ESBL-E in HT-29 enterocytes, and protected the enterocyte monolayers used in this study as a bio-model of the intestinal barrier. BLLT1 increased intestinal alkaline phosphatase (IAP) as one of the main molecular factors providing intestinal homeostasis. Conclusions: BLLT1 shows promise for the creation of innovative functional nutritional products for humans and feed additives for farm animals that will reduce the spread of ESBL-E strains in the food chain. Full article

Pullorum disease, an intestinal disease in chickens caused by Salmonella enterica serovar pullorum (S. Pullorum), is a significant threat to the poultry industry and results in substantial economic losses. The bacteria’s transmission, both vertical and horizontal, makes it difficult to completely eliminate it. Control strategies for pullorum disease primarily involve stringent eradication programs that cull infected birds and employ antibiotics for treatment. However, eradication programs are costly, and antibiotic use is restricted. Therefore, developing alternative control strategies is essential. Increasingly, studies are focusing on modulating the gut microbiota to control intestinal diseases. Modulating the chicken gut microbiota may offer a novel strategy for preventing and controlling pullorum disease in poultry. However, the impact of S. Pullorum on the chicken gut microbiota has not been well established, prompting our exploration of the relationship between S. Pullorum and the chicken gut microbiota in this study. In this study, we initially analyzed the dynamic distribution of the gut microbiota in chickens infected with S. Pullorum. Alpha diversity analysis revealed a decrease in observed OTUs and the Shannon diversity index in the infected group, suggesting a reduction in the richness of the chicken gut microbiota due to S. Pullorum infection. Principal coordinate analysis (PCoA) showed distinct clusters between the gut microbiota of infected and uninfected groups, indicating S. Pullorum infection changed the chicken gut microbiota structure. Specifically, S. Pullorum infection enriched the relative abundance of the genera Escherichia-Shigella (65% in infected vs. 40.6% in uninfected groups) and Enterococcus (10.8% vs. 3.7%) while reducing the abundance of Lactobacillus (9.9% vs. 32%) in the chicken microbiota. Additionally, based on the observed changes in the chicken gut microbiota, we isolated microorganisms, including Bifidobacterium pseudolongum, Streptococcus equi and Lacticaseibacillus paracasei (L. paracasei), which were decreased by S. Pullorum infection. Notably, the L. paracasei Lp02 strain was found to effectively inhibit S. Pullorum proliferation in vitro and alleviate its infection in vivo. We found that S. Pullorum infection reduced the richness of the chicken gut microbiota and enriched the relative abundance of the genera Escherichia-Shigella and Enterococcus while decreasing the abundance of the anaerobic genus Lactobacillus. Furthermore, microbiota analysis enabled the isolation of several antimicrobial microorganisms from healthy chicken feces, with a L. paracasei strain notably inhibiting S. Pullorum proliferation in vitro and alleviating its infection in vivo. Overall, this research enhances our understanding of the interaction between gut microbiota and pathogen infection, as well as offers new perspectives and strategies for modulating the chicken gut microbiota to control pullorum disease. Full article

Enterococcus species are significant intestinal commensals of animals, including poultry. However, they have emerged as important opportunistic infective agents in both veterinary and human medicine as well as major nosocomial pathogens, owing to their increasing antimicrobial resistance. This research aimed to investigate the prevalence and antimicrobial resistance patterns of Enterococcus spp. isolated from poultry farms in the north of Serbia. A total of 40 samples of overshoes or feces were collected from 40 poultry farms and analyzed for the presence of Enterococcus spp. using PCR or MALDI-TOF mass spectrometry for their identification. The number of isolates was 40 and included 11 isolates from laying hens, 2 isolates from turkeys, 3 from broiler breeders, and 24 from broilers. The Kirby–Bauer disk diffusion method was used to test for antibiotic susceptibility in accordance with the Clinical and Laboratory Standards Institute and EUCAST guidelines. The results showed that Enterococcus faecalis was isolated from 37.5% farms, and E. faecium from 42.5%. E. hirae was identified in 15% of poultry establishments, and E. durans and E. thialandicus on 2.5%. Notably, resistance to erythromycin, streptomycin, fluoroquinolones, and tetracyclines among the frequently used antibiotics was found. Furthermore, 35% of the isolates had multidrug resistance (MDR). In order to prevent the spread of antibiotic resistance in chicken farming and protect the health of the public and animals alike, our findings highlight the critical need for improved surveillance and control measures. To effectively establish a containment strategy for Enterococcus spp. isolated from poultry farms, more research into the processes behind their antibiotic resistance is required. Full article

Rapid growth in commercial poultry production is one of the major sources of Salmonella infections that leads to human salmonellosis. The two main Salmonella enterica serovars associated with human salmonellosis are enteritidis and typhimurium. The aim of this study was to determine the prevalence of S. enterica serovars Enteritidis and S. Typhimurium as well as their Salmonella pathogenicity islands (SPI) and antibiotic resistance profiles in broiler chicken feces from slaughterhouses. A total of 480 fecal samples from broiler chickens that were grouped into 96 pooled samples were identified to have Salmonella spp. using the invA gene, whilst the Spy and sdfI genes were used to screen for the presence of S. Enteritidis and S. Typhimurium serovars, respectively, by polymerase chain reaction (PCR) assays. The isolates were also screened for the presence of Salmonella pathogenicity islands (SPIs) using PCR. The disc diffusion assay was performed to determine the antibiotic resistance profiles of the isolates. A total of 36 isolates were confirmed as Salmonella spp. through amplification of the invA gene. Out of 36 confirmed Salmonella spp. a total of 22 isolates were classified as S. Enteritidis (n = 8) and were S. Typhimurium (n = 14) serovars. All (n = 22) S. Enteritidis and S. Typhimurium isolates possessed the hilA (SPI-1), ssrB (SPI-2) and pagC (SPI-11) pathogenicity islands genes. Amongst these serovars, 50% of the isolates (n = 11/22) were resistant to tetracycline and nalidixic acid. Only 22% of the isolates, S. Typhimurium (13.6%) and S. Enteritidis (9.1%) demonstrated resistance against three or more antibiotic classes. The most detected antibiotic resistance genes were tet(K), mcr-1, sulI and strA with 13 (59.1%), 9 (40.9%), 9 (40.9%) and 7 (31.8%), respectively. The findings of this study revealed that S. Typhimurium is the most prevalent serotype detected in chicken feces. To reduce the risk to human health posed by salmonellosis, a stringent public health and food safety policy is required. Full article

Although carbapenems have not been approved for animal use, carbapenem-resistant Escherichia coli (CREC) strains are increasingly being detected in food-producing animals, posing a significant public health risk. However, the epidemiological characteristics of CREC isolates in yellow-feather broiler farms remain unclear. We comprehensively investigated the genetic features of carbapenem-resistance genes among E. coli isolates recovered from a yellow-feather broiler farm in Guangdong province, China. Among the 172 isolates, 88 (51.2%) were recovered from chicken feces (88.5%, 54/61), the farm environment (51.1%, 24/47), and specimens of dead chickens (15.6%, 41/64). All CREC isolates were positive for the bla_NDM-5 gene and negative for other carbapenem-resistance genes. Among 40 randomly selected isolates subjected to whole-genome sequencing, 10 belonged to distinct sequence types (STs), with ST167 (n = 12) being the most prevalent across different sources, suggesting that the dissemination of bla_NDM-5 was mainly due to horizontal and clonal transmission. Plasmid analysis indicated that IncX3, IncHI2, and IncR-X1-X3 hybrid plasmids were responsible for the rapid transmission of the bla_NDM-5 gene, and the genetic surrounding of bla_NDM-5 contained a common mobile element of the genetic fragment designated “IS5-△ISAba125-bla_NDM-5-ble_MBL-trpF-dsbC”. These findings demonstrate a critical role of multiple plasmid replicons in the dissemination of bla_NDM-5 and carbapenem resistance. Full article