Intestinal Isolates Research Articles

Sequence Types and Antimicrobial Resistance Profiles of Salmonella Typhimurium in the Food Chain in Singapore.

Salmonella remains a significant foodborne pathogen globally with S. Typhimurium presenting as a frequently occurring serovar. This study aimed to characterize 67 S. Typhimurium isolates from humans, food, farms, and slaughterhouses collected in Singapore from 2016 to 2017. Using whole-genome sequencing analysis, the isolates were found to belong to either ST19 (n = 33) or ST36 (n = 34). ST36 predominated in human intestinal and chicken isolates, while human extra-intestinal and non-chicken food isolates belonged to ST19. Plasmids were predicted in 88.1% (n = 59) of the isolates with the most common incompatibility group profiles being IncFIB(S), IncFII(S) and IncQ1. IncFIB(S) (adjusted p-value < 0.05) and IncFII(S) (adjusted p-value < 0.05) were significantly more prevalent in ST19 isolates, while Col156 (adjusted p-value < 0.05) was more significantly found in ST36 isolates. ST36 isolates exhibited higher resistance to multiple antibiotic classes such as penicillins, phenicols, folate pathway inhibitors, aminoglycosides, β-lactam/β-lactamase inhibitor combinations, tetracyclines, and fluoroquinolones. Phylogenetics analysis suggested potential shared routes of transmission among human, chicken, farm and slaughterhouse environments. Taken together, this study offers a cross-sectional epidemiological insight into the genomic epidemiology and antimicrobial landscape of S. Typhimurium isolates in Singapore, informing strategies for future public health and food safety surveillance.

Isolate distribution and antifungal susceptibility of Saccharomyces cerevisiae in the national regional medical center of Southwest China for women and children during 2018–2023

BackgroundSaccharomyces cerevisiae has been considered a harmless yeast, but in recent years, increasing evidence has shown that it can cause disease in humans, especially invasive infections in infants/children and vulvovaginal infections in women. This study aimed to investigate the clinical information and antifungal susceptibility of clinical cases with S. cerevisiae and establish a foundation for the prevention and treatment of fungal infections.MethodsThis study was conducted from May 2018 to May 2023 at a national regional medical center in Southwest China for women and children. The demographic and clinical characteristics of patients isolated with S. cerevisiae were collected and analyzed. All the isolates were cultured on Sabouraud medium plates and identified by MALDI-TOF MS. The antifungal susceptibility of S. cerevisiae to 10 agents (amphotericin B, fluconazole, itraconazole, voriconazole, micafungin, caspofungin, terbinafine and 5-flucytosine) was determined via the microdilution broth method to determine the minimum inhibitory concentrations (MICs).ResultsA total of 75 cases of S. cerevisiae isolated from patients with vulvovaginal candidiasis (VVC, 44 cases), pneumonia (13 cases), or diarrhea (18 cases) were included after data review. The MICs of voriconazole and flucytosine for S. cerevisiae isolated from different body sites differed, with higher resistance in intestinal isolates. In this study, S. cerevisiae caused VVC, but there was no clear evidence that it was involved in pneumonia or diarrhea. Compared with those of Candida albicans, the primary pathogen of VVC, the MICs of fluconazole (11.96 ± 5.78 µg/mL vs. 67.64 ± 16.62 µg/mL, p = 0.002), itraconazole (0.77 ± 0.19 µg/mL vs. 2.31 ± 0.53 µg/mL, p = 0.008), voriconazole (0.22 ± 0.09 µg/mL vs. 5.02 ± 1.09 µg/mL, p < 0.001), and terbinafine (10.41 ± 0.84 µg/mL vs. 14.93 ± 4.77 µg/mL, p < 0.001) for S. cerevisiae (isolated from the genital tract) were significantly lower, while those of micafungin (0.14 ± 0.01 µg/mL vs. 0.06 ± 0.01 µg/mL, p < 0.001) and caspofungin (0.27 ± 0.04 µg/mL vs. 0.06 ± 0.01 µg/mL, p < 0.001) were significantly greater.ConclusionAzoles remain the recommended regimen for S. cerevisiae-related VVC, and the use of amphotericin B vaginal effervescent tablets could be considered for the treatment of azole-resistant isolates. The antifungal susceptibility of S. cerevisiae varies according to the isolated source, and the pathogenicity trend of S. cerevisiae should be studied.

Non-lactic acid bacteria probiotics isolated from intestine or various circumstances.

Probiotics are live microorganisms beneficial to host health, predominantly comprising lactic acid bacteria (LAB) such as Lactobacillus. Additional non-LAB probiotics, termed intestinal isolates, encompass next-generation strains like Akkermansia muciniphila, Faecalibacterium prausnitzii, Christensenella minuta, Anaerobutyricum soehngenii, Oxalobacter formigenes, etc. and alongside externally sourced Bacillus, Saccharomyces cerevisiae, Clostridium butyricum, and Propionibacterium. Intestinal-derived probiotics represent strictly anaerobic strains with challenging culturing requirements, contrasting with the aerobic nature of Bacillus probiotics and the ease of culturing S. cerevisiae. These strains exhibit diverse health-promoting properties, encompassing antimicrobial, anticancer, antioxidant, and vitamin production capabilities, albeit contingent upon strain specificity. This review delineates the characteristics, culturing conditions, and health advantages associated with non-LAB probiotics.

Pengaruh Pemberian Bakteri dan Trichoderma viride dari Isolat Usus Larva Black Soldier Fly (BSF) terhadap Ketahan Penyakit Cabai Keriting (Capsicum annuum)

Capsicum annuum (curly red chili) is an important commodity for the community. The addition of chemical pesticides is used by farmers to increase plant disease resistance. The use of chemical pesticides is harmful to the environment and can have long-term effects if consumed. Therefore, biopesticide innovation is needed. Black Soldier Fly (BSF) is an insect that has bacteria and fungi in its gut. Bacteria and fungi found in BSF intestinal isolates can function as antimicrobials for plants. Because of this, in this study investigated the effect of the bacteria and fungi Trichoderma viride in increasing disease resistance in C. annuum plants. There were 4 treatments in this study, namely the administration of chemical fungicides, BSF bacterial formula, T. viride. BSF, and a mixture of bacteria and T. viride BSF. The results obtained were analyzed by calculating the Intensity of Attack (IS) score, microscopic observations in the laboratory, and statistical tests. The IS score of the control plants was 75.0%, the fungicide treatment was 37.5%, the bacterial treatment alone was 33.3%, the T. viride treatment was 19.4%, and the mixed treatment of bacteria and T. viride was 4.2%. All plants were attacked by leaf spot disease by Cercospora sp. Control plants were attacked by Sclerotium rolfsii fungus wilt and powdery mildew by Leveillula taurica and jaundice. Jaundice also attacked plants treated with T. viride only and bacteria only. The normality and homogeneity tests show that the data is not normal and not homogeneous but H1 is acceptable based on the Kruskal Wallis test. The conclusion of this study is that bacteria and T. viride from BSF are able to increase disease resistance in curly chili plants, but are not yet effective in preventing disease caused by Cercospora sp.

Regulation of intestinal flora in patients with chronic atrophic gastritis by modified Chai Shao Liu Jun Zi decoction based on 16S rRNA sequencing.

Chai Shao Liu Jun Zi decoction (CSLJZD) is an effective Chinese medicine for the treatment of chronic atrophic gastritis (CAG). However, the effect of CSLJZD on the intestinal flora of patients with CAG remains unclear. We used 16S rRNA gene sequencing to investigate the regulatory effects of CSLJZD on intestinal microflora in patients with CAG. Eight patients with CAG were randomly selected as the model group and 8 healthy medical examiners as the control group; the treatment group comprised patients with CAG after CSLJZD treatment. High-throughput sequencing and bioinformatics analysis of the V3V4 region of the 16S rRNA gene of intestinal bacteria obtained from the intestinal isolates of fecal specimens from all participants were performed separately. A rarefaction curve, species accumulation curve, Chao1 index, and ACE index were calculated to assess the alpha diversity. Principal component analysis (PCA), non-metric multi-dimensional scaling, and the unweighted pair group method with arithmetic mean were used to examine beta diversity. The LEfSe method was used to identify the differentially expressed bacteria. Differential function analysis was performed using PCA based on KEGG function prediction. Rarefaction and species accumulation curves showed that the sequencing data were reasonable. The Chao1 and ACE indices were significantly increased in patients with CAG compared with those in the healthy group. Following CSLJZD and vitacoenzyme treatment, Chao1 and ACE indices decreased. The PCA, non-metric multi-dimensional scaling, and unweighted pair group method with arithmetic mean results showed that the CAG group was distinct from the healthy and treatment groups. The LEfSe results showed that the abundances of the genus Bilophila, family Desulfovibrionaceae, order Desulfovibrionales and genus Faecalibacterium were significantly higher in the healthy group. The abundance of genus Klebsiella, order Deltaproteobacteria, genus Gemmiger, and other genera was significantly higher in the treatment group. Treatment with CSLJZD had a therapeutic effect on the intestinal flora of patients with CAG.

Biofilm Formation in Campylobacter concisus: The Role of the luxS Gene.

Campylobacter concisus is a bacterium that inhabits human oral cavities and is an emerging intestinal tract pathogen known to be a biofilm producer and one of the bacterial species found in dental plaque. In this study, biofilms of oral and intestinal C. concisus isolates were phenotypically characterized. The role of the luxS gene, which is linked to the regulation of biofilm formation in other pathogens, was assessed in relation to the pathogenic potential of this bacterium. Biofilm formation capacity was assessed using phenotypic assays. Oral strains were shown to be the highest producers. A luxS mutant was created by inserting a kanamycin cassette within the luxS gene of the highest biofilm-forming isolate. The loss of the polar flagellum was observed with scanning and transmission electron microscopy (SEM and TEM). Furthermore, the luxS mutant exhibited a significant reduction (p < 0.05) in biofilm formation, motility, and its expression of flaB, in addition to the capability to invade intestinal epithelial cells, compared to the parental strain. The study concluded that C. concisus oral isolates are significantly higher biofilm producers than the intestinal isolates and that LuxS plays a role in biofilm formation, invasion, and motility in this bacterium.

Diversity analysis of hilsa (Tenualosa ilisha) gut microbiota using culture-dependent and culture-independent approaches.

The bacterial communities associated with the gastrointestinal tract are primarily involved in digestion, physiology and immune response against pathogenic bacteria for the overall development and health of the host. Hilsa shad (Tenualosa ilisha), a tropical anadromous fish, found predominantly in Bangladesh and India, has so far been poorly investigated for its gut bacterial communities. In this study, both culture-based and metagenomic approaches were used to detect intestinal isolates of hilsa, captured from both freshwater and seawater to investigate the community structure of intestinal microbiota. Culture-dependent approach allowed to isolate a total of 23 distinct bacterial species comprising sixteen Gram-negative, and seven Gram-positive isolates, where Proteobacteria and Firmicutes were identified as the two most dominant phyla. While metagenomic approach explored a wide range of important gastrointestinal bacteria, primarily dominated by Proteobacteria, Firmicutes and Bacteroidetes, with Proteobacteria and Firmicutes, being the most abundant in freshwater and seawater samples, respectively. A combination of these approaches provided the differential gastrointestinal-associated bacterial diversity in freshwater and seawater hilsa with the prediction of overall functional potential.

Detection and Characterization of hly,cdtI and stxI genes, in MDR uropathogenic and intestinal E.coli isolates, Hilla city, Iraq.

Background:. Escherichia coli infections represent a considerable burden in human. Illness caused by E. coli have a significant burden on patients and the healthcare system, so prompt recognition, and appropriate treatment are necessary. Escherichia coli is the first bacterial pathogen in humans responsible for deaths associated with antibiotic resistance&#x0D; Aim of study: detection of hly, cdtI and stxI genes through molecular analysis in MDR E.coli isolates.&#x0D; Methods: From November 2021 to May 2022, atotal of 150 urine samples were collected from patients suffering from urinary tract infections were admitted and visit Maternity and Children Babylon Hospital in Al-Hilla city. 100 stool samples were (as control). The identification and antibiotic susceptibility profile of E.coli were done by vitek2 compact system.The isolates were subjected to PCR assays with specific primers for hly , cdtI and stxI .&#x0D; Results: Only 50(45.5%) E.coli isolates were recovered from the urine samples. In contrast, 60(60%) E.coli isolates from the stool samples. A high rate of MDR was detected among E. coli isolates, where it was( 60%) and (53.3%) in the uropathogenic and intestinal isolates, respectively.&#x0D; The PCR results were showed that MDR uropathogenic E. coli and intestinal E.coli were hardbourd hly gene (46.7%)and (90.6%), respectively. The cdtI gene was detected in (43.4%) of uropathogenic E. coli, and (40.6%) of intestinal E.coli. The stx gene was detected in (16.7%) of uropathogenic E. coli, and (43.7%) of intestinal E.coli.&#x0D; Conclusion: The presence of a high percentage of toxin in intestinal E.coli is a serious warning because of the possibility of their becoming virulent if the bacteria change their natural habitat or are exposed to a certain stress.

Population dynamics and characteristics of Klebsiella pneumoniae from healthy poultry in Norway.

Klebsiella pneumoniae is an important opportunistic pathogen widely studied in relation to human infection and colonization. However, there is a lack of knowledge regarding other niches that K. pneumoniae may inhabit. K. pneumoniae isolated from healthy broiler and turkey flocks in Norway in 2018 have previously been described with regard to population structure, sequence types (STs), and the presence of virulence- and antimicrobial resistance (AMR) genes. In the present study we aimed to evaluate the dynamics of the K. pneumoniae population in poultry over time, with regards to AMR and virulence, and with a special focus on persistence of STs. A total of 391 flocks sampled in 2020 were included in the present study, of which 271 were from broiler flocks and 120 from turkey flocks. Similar to findings from 2018, the occurrence of K. pneumoniae was significantly higher based on culturing in turkey flocks (62.5%) compared to broiler flocks (24.0%). Major STs in 2020 included ST5827 (n = 7), ST37 (n = 7), ST370 (n = 7), ST17 (n = 5), and ST4710 (n = 5). Several STs persisted over time in both host species, including ST35, ST37, ST590, and ST17. This persistence may be due to local re-circulation or reintroduction from parent flocks. Of these five major STs, only ST590 carried AMR genes, indicating that the persistence was not associated with the presence of AMR genes. An ST4710 strain with a hypervirulence-encoding plasmid (p4710; iro5, iuc5) was recovered from turkeys in 2018. The same strain was present in turkeys in 2020, but the plasmid had lost the salmochelin locus. This loss may be attributed to reductive evolution due to the presence of several siderophores within the same isolates. In this study we also characterized a clinical ST4710 isolate from a turkey with airsacculitis. The isolate was closely related to two intestinal ST4710 isolates from healthy turkeys in 2018. These three isolates were sampled within the same location and time frame in 2018, and all carried the full p4710 virulence plasmid. These findings highlight the transmission- and infectious potential of ST4710 in turkeys.

An Intestinal Bacillus velezensis Isolate Displays Broad-Spectrum Antibacterial Activity and Prevents Infection of Both Gram-Positive and Gram-Negative Pathogens In Vivo.

The increasing prevalence of drug-resistant bacteria has significantly diminished the effectiveness of antibiotics in clinical settings, leading to the emergence of untreatable bacterial infections. To address this public health challenge, the gut microbiome represents a promising source of novel antimicrobial therapeutics. In this study, we screened mouse intestinal isolates for growth inhibitory activity against the human enteric pathogen Vibrio cholerae and identified a strain of spore-forming Bacillus velezensis, named BVM7, that produced a potent antibiotic with activity against V. cholerae and a broad spectrum of enteric and opportunistic pathogens. Characterization of the antimicrobial compounds produced by BVM7 revealed that they were primarily secreted antimicrobial peptides (AMPs) produced during stationary-phase growth. Furthermore, our results showed that introducing either BVM7 vegetative cells or spores into mice precolonized with V. cholerae or Enterococcus faecalis significantly reduced the burden of infection. Interestingly, we also observed that BVM7 was sensitive to a group of Lactobacillus probiotic strains and that inoculation of Lactobacilli could eliminate BVM7 and potentially restore the native gut microbiome. These findings highlight the potential of bacteria from the gut microbiome as a source for novel antimicrobial compounds and a tool for managing bacterial infections by in situ bio-delivery of multiple AMPs. IMPORTANCE The rise of antibiotic-resistant pathogens poses a challenge to public health. The gut microbiome presents a promising source of new antimicrobials and treatments. By screening murine gut commensals, we found a spore-forming Bacillus velezensis strain, BVM7, that exhibited antimicrobial activity toward a wide array of enteric and opportunistic bacterial pathogens. In addition to showing that this killing effect occurred through the action of secreted antimicrobial peptides (AMPs), we demonstrate that BVM7 vegetative cells and spores can be used to treat infections of both Gram-positive and Gram-negative pathogens in vivo. By expanding our knowledge of the antimicrobial properties of bacteria in the gut microbiome, we hope to contribute insights for developing novel drugs and therapeutic interventions.

Profiling and antibiotic resistance of some human intestinal isolates

The cases of antibiotic resistance in Enterobacteriaceae are of great global concern. This study aimed at contributing towards the fight against antibiotic resistance and ameliorate the management/treatment of Enterobacteriaceae-linked diseases. Ten (10) rectal swab samples were collected from five (5) male and five (5) females and two isolates were identified from the samples collected. The isolates were identified through colonial, morphological and biochemical tests carried out following standard procedures. Isolates were investigated for their antibiotic resistance profile, Multiple Antibiotic Resistance indices (MARi), pathogenicity and their resistance genes were identified through molecular means using plasmid amplification and primers. Primers used include. ermB, BlaTem, qnrB genes Result obtained showed the isolates to be Escherichia coli and Klebsiella oxytoca. E. coli showed Alpha (α) heamolysis, while Klebsiella oxytoca showed gamma (γ) heamolysis . E. coli was resistant to 75% of the antibiotics used, Klebsiella oxytoca was sensitive to 42% of all the antibiotics. All the test organisms were resistant to all classes of Cephalosporins. The plasmid profiling revealed that all isolates have low molecular weight plasmids. The molecular fingerprinting of the isolates using gene primers viz -a viz ermB, BlaTem, qnrB genes showed E. coli to have resistance genes for macrolides (ermB gene) while none of the isolates had resistance factor against quinolones (qnrB gene). This study showed a high carriage of Enterobacteriaceae having phenotypic resistance with corresponding plasmid-borne resistance genes. The need to understand how bacteria adapt to the antibiotic environment will lead to new therapeutic strategies for antibiotic-resistant infections. Interventions measures to minimize the abundance of antibiotic-resistant commensals and opportunistic pathogens may include faecal microbiota transplantation and the use of live biotherapeutics.

Prevalence and molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus in the respiratory tracts of Chinese adults with community-acquired pneumonia

BackgroundCommunity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an important pathogen causing healthcare-associated infections. In recent years, an increasing number of CA-MRSA clones have emerged and rapidly spread in the community and hospital settings in China. ObjectivesTo investigate the molecular epidemiology and resistance of CA-MRSA in the respiratory tracts of Chinese adults with community-acquired pneumonia (CAP). MethodsA total of 243 sputum samples were collected from adult patients with CAP at the Nantong Hospital in China between 2018 and 2021. S. aureus was identified using PCR, and its susceptibility to 14 antimicrobials was tested using the broth dilution method. Genomic characterization of respiratory CA-MRSA and our previously collected intestinal CA-MRSA isolates was performed using whole-genome sequencing, and the evolutionary relationships of these isolates were assessed using phylogenetic analysis. ResultsThe CA-MRSA colonization rate among adults with CAP in China was 7.8 % (19/243). Antimicrobial resistance analysis revealed that the proportion of multidrug-resistant respiratory CA-MRSA isolates (100 %) was higher than that of intestinal CA-MRSA isolates (6.3 %). Among the 35 CA-MRSA isolates, 10 MLST types were identified and clustered into five clone complexes (CCs). CC5 (48.6 %) and CC88 (20 %) were predominant CA-MRSA clones. Notably, the CC5 clone ST764/ST6292-MRSA-II-t002 was identified as the major lineage causing respiratory tract infections in Chinese adults with CAP. ConclusionsThe prevalence of CA-MRSA among Chinese adults with CAP is high and often involves ST764/ST6292-MRSA-II-t002 as the causal pathogen.

Genetic diversity of Siberian bovine coronavirus isolates (Coronaviridae: Coronavirinae: Betacoronavirus-1: Bovine-Like coronaviruses)

Bovine coronaviruses (BCoVs) are causative agents of diarrhea, respiratory diseases in calves and winter cow dysentery. The study of genetic diversity of these viruses is topical issue. The purpose of the research is studying the genetic diversity of BCoV isolates circulating among dairy cattle in Siberia. Specimens used in this study were collected from animals that died or was forcedly slaughtered before the start of the study. The target for amplification were nucleotide sequences of S and N gene regions. Based on the results of RT-PCR testing, virus genome was present in 16.3% of samples from calves with diarrheal syndrome and in 9.9% with respiratory syndrome. The nucleotide sequences of S gene region were determined for 18 isolates, and N gene sequences - for 12 isolates. Based on S gene, isolates were divided into two clades each containing two subclades. First subclade of first clade (European line) included 11 isolates. Second one included classic strains Quebec and Mebus, strains from Europe, USA and Korea, but none of sequences from this study belonged to this subclade. 6 isolates belonged to first subclade of second clade (American-Asian line). Second subclade (mixed line) included one isolate. N gene sequences formed two clades, one of them included two subclades. First subclade included 3 isolates (American-Asian line), and second subclade (mixed) included one isolate. Second clade (mixed) included 8 sequences. No differences in phylogenetic grouping between intestinal and respiratory isolates, as well as according to their geographic origin were identified. The studied population of BCoV isolates is heterogeneous. Nucleotide sequence analysis is a useful tool for studying molecular epidemiology of BCoV. It can be beneficial for choice of vaccines to be used in a particular geographic region.

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.

Detection of carbapenem resistant Escherichia coli in cattle and pigs

The aim of this study was to detect the presence of carbapenem resistant Escherichia coli (E. coli) in cattle and pigs. Altogether, 100 samples were collected, 50 each from cattle and pigs including mastitic milk and faecal samples from healthy as well as diarrhoeic cattle and faecal samples and slaughter specimens from pigs. Bacteria were isolated from the samples and antibiotic sensitivity test was carried out. Eight isolates of E. coli were obtained from cattle and 15 isolates from pigs. The phenotypic resistance patterns against carbapenems (ertapenem, imipenem and meropenem) were analysed. In cattle, the four isolates from milk were observed to be sensitive to all the three carbapenems whereas, all the isolates obtained from faecal samples were resistant to all the three carbapenems. The four faecal and three intestinal isolates of E. coli from pigs showed sensitivity to all the three carbapenems. From a total of 10 faecal samples from pigs, four, three and one isolates showed resistance to ertapenem, imipenem and meropenem respectively. Among five isolates from intestine, one isolate showed resistance to ertapenem and imipenem and no isolate showed resistance to meropenem. Polymerase chain reaction (PCR) was carried out to find the presence of carbapenem resistance genes (blaSHV, blaCTX, blaTEM and blaNDM) in all the 23 isolates and the results revealed that seven (58.3 per cent) E. coli isolates carried blaSHV gene and four (33.3 per cent) contained blaCTX gene which encodes carbapenem resistant enzymes. Antibiogram using 14 commonly used antimicrobial agents revealed varied multidrug resistant patterns among the E. coli isolates.

Prevalence of Genes Encoding Resistance to Aminoglycosides and Virulence Factors Among Intestinal Vancomycin-Resistant Enterococci

Background: Vancomycin-resistant enterococci (VRE) are recognized as nosocomial pathogens with increased importance in recent years. These bacteria are frequently isolated from patients admitted to intensive care units (ICUs). Enterococcal pathogenicity is enhanced by different antibiotic resistance and virulence determinants. Objectives: The present study aimed to assess the prevalence of genes encoding resistance to antibiotics and virulence factors in intestinal VRE isolates from ICU patients. Methods: In this study, 23 VREs were investigated. Minimum inhibitory concentrations (MICs) to nine antimicrobial agents were examined using E-test. Genes encoding vancomycin resistance (vanABCDMN), aminoglycoside-modifying enzymes (aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa, ant(3')-Ia, ant(4')-Ia, ant(6')-Ia), together with genes for various virulence factor (ace/acm, asa1, cylA, efaA, esp, gelE and hyl), were detected using multiplex PCR. Results: The species distribution of the tested VRE was as follows: Nine Enterococcus casseliflavus, seven E. gallinarum, and seven E. faecium. The vanA gene was found in all E. faecium, in six of which the classical VanA phenotype was observed. The vancomycin (vanC) phenotype was associated with the presence of vanC1 gene in E. gallinarum and the vanC2 gene in E. casseliflavus isolates. The aac(6')-Ie-aph(2")-Ia gene was encoding high-level gentamicin resistance (HLGR) in the studied VRE. All E. faecium were positive for acm and esp, while acm in combination with esp or hyl was detected in 2 vanC enterococci. Conclusions: According to the findings, there was a correlation between the phenotype and the genotype of glycopeptide resistance in the tested VRE. HLGR was more prevalent in E. faecium because of the presence of aac(6')-Ie-aph(2")-Ia. The higher prevalence of virulence determinants was confirmed in vanA isolates compared to the studied vanC-carrying enterococci.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool

ABSTRACT Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota. Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences

Shiga Toxin-Producing Escherichia coli in Diarrheic Neonatal Dairy Calves: Matching Small Intestinal and Fecal Isolates in Terms of Isolation Rates and Antimicrobial Resistance

Background: Shiga toxin-producing strains have been considered remarkable diarrheagenic agents and foodborne pathogens. Several studies have mentioned the role of some Shiga toxin-producing Escherichia coli (STEC) strains in diarrhea and dysentery in calves. Enteropathogenic E. coli (EPEC) have also been isolated from diarrheic calves. Generally, the culture and antibiogram results obtained from fecal samples are used to select antibiotics to treat calf diarrhea. However, the value of such a sampling method has not been evaluated yet. Objectives: This study aimed to evaluate the clinical utility of fecal sample cultures for isolating STEC and EPEC in calf diarrhea by comparing them with small intestine samples. Materials and Methods: The small intestine and fecal samples were simultaneously collected from 35 diarrheic calves. Small intestine samples were collected under the ultrasonographic guide. A total of 70 confirmed E. coli isolates were screened by the multiplex polymerase chain reaction to detect genes encoding Shiga toxin1 (stx1), Shiga toxin2 (stx2), intimin (eae), and hemolysin (E-hly). We also compared the presence of class 1 and 2 integrons and antimicrobial resistance properties in the STEC and EPEC isolates recovered from the small intestine and fecal samples. Finally, the presence of important STEC/EPEC serogroups, including O26, O103, O111, O113, O145, and O157 in isolates from both samples, was determined as well. Results: STEC strains were detected in 25.7%, and 20% of E. coli isolates obtained from the small intestine and fecal samples, respectively. The stx1 was the sole Shiga toxin subtype detected among STEC in intestinal and fecal isolates. EPEC was detected only in one and two E. coli isolates from the small intestine and fecal samples, respectively. Conclusion: A numerically higher prevalence of STEC was observed in the small intestine compared to fecal samples; there was no significant difference in the frequencies of STEC and EPEC isolates between the small intestine and fecal samples. The results indicated that the fecal sample, as a non-invasive and practical method, could be used for isolating STEC and EPEC in calf diarrhea. The antibiogram showed the presence of a high degree of multi-drug resistance among the isolates.

Black soldier fly larvae effectively degrade lincomycin from pharmaceutical industry wastes

Lincomycin fermentation residues (LFR) are the byproducts from the pharmaceutical industry, and contain high concentrations of antibiotics that could pose a threat to the environment. Here, we report that black soldier fly larvae (BSFL) and associated microbiota can effectively degrade LFR and accelerate the degradation of lincomycin in LFR. The degradation rate of lincomycin in LFR can reach 84.9% after 12 days of BSFL-mediated bioconversion, which is 3-fold greater than that accomplished with natural composting. The rapid degradation was partially carried out by the BSFL-associated microbiota, contributing 22.0% of the degradation in the final composts. Based on microbiome analysis, we found that the structure of microbiota from both BSFL guts and BSFL composts changed significantly during the bioconversion, and that several bacterial genera were correlated with lincomycin degradation. The roles of the associated microbiota in the degradation were further verified by the ability of two larval intestinal bacterial isolates and one bacterial isolate from BSFL composts to lincomycin degradation. The synergy between BSFL and the isolated strains resulted in a 2-fold increase in degradation compared to that achieved by microbial degradation alone. Furthermore, we determined that the degradation was correlated with the induction of several antibiotic resistant genes (ARGs) associated with lincomycin degradation in larval guts and BSFL composts. Moreover, the environmental conditions in the BSFL composts were found to be conducive to the degradation. In conclusion, these findings demonstrate that the BSFL-mediated bioconversion of LFR could effectively reduce residual lincomycin and that the associated microbiota play crucial roles in the process.

Comparison of Carbapenem-Resistant Klebsiella pneumoniae Strains Causing Intestinal Colonization and Extraintestinal Infections: Clinical, Virulence, and Molecular Epidemiological Characteristics

Carbapenem-resistant Klebsiella pneumonia (CRKP) infections has become a concerning threat. However, knowledge regarding the characteristics of intestinal CRKP isolates is limited. This study aimed to investigate and compare the clinical, virulence and molecular epidemiological characteristics of intestinal colonization and extraintestinal infections CRKP strains. The clinical characteristics were investigated retrospectively. Polymerase chain reaction was used to investigate the capsular serotype, virulence genes and carbapenemase genes. Capsular polysaccharide quantification assay, serum resistance assay, biofilm formation assay, and infection model of Galleria mellonella larvae were performed to compare the virulence and pathogenicity. Besides, multilocus-sequence-typing (MLST) and pulsed-field-gel-electrophoresis (PFGE) were conducted to explore the homology of intestinal CRKP isolates. A total of 54 intestinal CRKP isolates were included. The main capsular serotypes were K14, K64, and K19. C-reactive protein and the proportion of ICU isolation of the infection group were significantly higher than that of the colonization group (P < 0.05). The carrier rates of various virulence genes of CRKP in the infection group were mostly higher than those in the colonization group, wherein the carrier rates of peg-344 and rmpA were significantly different (P < 0.05). There was no significant difference in capsular polysaccharides, antiserum ability, biofilm formation ability between the two group (P > 0.05), but the lethality of the infection group to Galleria mellonella was significantly higher than that of the colonization group (P < 0.05). The MLST categorized the 54 isolates into 13 different sequence types. PFGE revealed that homology among the 54 CRKP strains was <80%. This study suggested that the CRKP strains in the infection group had higher virulence than those in the colonization group. The development of CRKP isolates colonizing in the intestine should be addressed in future clinical surveillance.