Colistin Resistance in Multidrug-Resistant Escherichia coli Isolated from Retail Broiler Meat in Bangladesh.

The emergence of colistin resistance in Escherichia coli is a global public health concern. Contaminated food can accelerate the spread of colistin-resistant E. coli to humans. This study aimed to detect and characterize colistin-resistant E. coli from broiler meat in Bangladesh. We analyzed 136 pooled broiler meat samples from 240 carcasses collected from 40 live bird markets in urban and rural areas and 8 metropolitan supermarkets. The mean count of E. coli in broiler meat samples collected from rural retail shops, metropolitan supermarkets, and urban retail shops was 5.3 ± 1.1, 4.1 ± 1.4, and 3.9 ± 0.8 log10 colony-forming unit per gram, respectively. Colistin-resistant E. coli (minimum inhibitory concentration >2 mg/L) was found in 78% (95% confidence interval 70.2-84.1%) of the samples. All colistin-resistant isolates harbored the mcr-1 gene, while the rest of the mcr genes (mcr-2 to mcr-9) were not detected. Most colistin-resistant E. coli isolates (98%) showed coresistance to tetracycline, sulfamethoxazole/trimethoprim followed by ciprofloxacin (95%). Alarmingly, all of the colistin-resistant isolates were found to be multidrug resistant. Phylogenetic analysis showed close similarities of the mcr-1 gene sequences of this study with many strains of Enterobacterales isolated from humans, animals, and the environment. This study detected colistin-resistant E. coli contamination in broiler meat, which can pose a serious public health threat.

Relatively preserved functional immune capacity with standard COVID-19 vaccine regimen in people living with HIV.

Introduction: People living with HIV (PLWH) are at a higher risk of severe disease with SARS-CoV-2 virus infection. COVID-19 vaccines are effective in most PLWH. However, suboptimal immune responses to the standard two-shot regimen are a concern, especially for those with moderate to severe immunodeficiency. An additional dose is recommended as part of the extended primary series in Taiwan. Herein, we study the efficacy of this additional shot in PLWH with mild immunodeficiency compared to that in healthy non-HIV people. Methods: In total, 72 PLWH that were asymptomatic or with mild immunodeficiency (CD4 counts ≥200/mm3) and suppressed virology, and 362 healthcare workers of our hospital were enrolled. None of the participants had a history of SARS-CoV-2 infection. They received mRNA-1273 and ChAdOx1 vaccines. Anti-SARS-CoV-2 neutralizing and anti-Spike IgG antibodies, and SARS-CoV-2-specific T cell responses were evaluated. Results: The standard two-shot regimen elicited lower responses in PLWH than the healthcare workers without HIV infection, although the difference was statistically insignificant. They had comparable levels of neutralizing and anti-Spike antibodies and comparable effector CD4+ and CD8+ T cell responses. The third shot boosted the SARS-CoV-2 immunity significantly more with better antibody responses and higher IFN-γ and IL-2 responses of the CD4+ and CD8+ T cells in PLWH compared to those without HIV. Upon in vitro stimulation with extracted Wuhan strain SARS-CoV-2 proteins, CD8+ T cells from PLWH after 3 shots had more durable effector responses than the non-HIV controls with extended time of stimulation. Conclusion: This subtle difference between PLWH and non-HIV people implied immune exhaustion with two shots in non-HIV people. Slightly compromised immunity in PLWH indeed preserved the functional capacity for further response to the third shot or natural infection.

Impact of antimicrobial use on abundance of antimicrobial resistance genes in chicken flocks in Vietnam.

Objectives: We investigated longitudinally Vietnamese small-scale chicken flocks in order to characterize changes in antimicrobial resistance gene (ARG) content over their life cycle, and the impact of antimicrobial use (AMU) on an intervention consisting of veterinary advice provision. Methods: AMU data and faecal samples were collected from 83 flocks (25 farms) at day-old, mid- and late-production (∼4 month cycle). Using high-throughput real-time PCR, samples were investigated for 94 ARGs. ARG copies were related to 16S rRNA and ng of DNA (ngDNA). Impact of AMU and ARGs in day-olds was investigated by mixed-effects models. Results: Flocks received a mean (standard error, SE) animal daily dose (ADD) of 736.7 (83.0) and 52.1 (9.9) kg in early and late production, respectively. Overall, ARGs/16S rRNA increased from day-old (mean 1.47; SE 0.10) to mid-production (1.61; SE 0.16), further decreasing in end-production (1.60; SE 0.1) (all P > 0.05). In mid-production, ARGs/16S rRNA increased for aminoglycosides, phenicols, sulphonamides and tetracyclines, decreasing for polymyxins ß-lactams and genes that confer resistance to mutiple classes (multi-drug resistance) (MDR). At end-production, aminoglycoside resistance decreased and polymyxin and quinolone resistance increased (all P < 0.05). Results in relation to ngDNA gave contradictory results. Neither AMU nor ARGs in day-olds had an impact on subsequent ARG abundance. The intervention resulted in 74.2% AMU reduction; its impact on ARGs depended on whether ARGs/ngDNA (+14.8%) or ARGs/16S rRNA metrics (-10.7%) (P > 0.05) were computed. Conclusions: The flocks' environment (contaminated water, feed and residual contamination) is likely to play a more important role in transmission of ARGs to flocks than previously thought. Results highlight intriguing differences in the quantification of ARGs depending on the metric chosen.

An IL-17-EGFR-TRAF4 axis contributes to the alleviation of lung inflammation in severe influenza.

Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. Our results suggest that viral neuraminidase-activated TGF-ß of the Th1 cells guides the Th17 evolution, and IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

Targeting influenza A virus by splicing inhibitor herboxidiene reveals the importance of subtype-specific signatures around splice sites.

BACKGROUND: The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene. METHODS: We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance. RESULTS: M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels. CONCLUSIONS: The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target.

The origin of regulatory from the effector cells in LAG-3-marked Th1 immunity against severe influenza virus infection.

In severe respiratory virus infections, including influenza, an exaggerated host immune response has been linked to the severe disease and death. Control of the overwhelming immune response is thus essential. Efforts with broad-spectrum immunosuppressive agents such as steroids are disappointing. A better understanding of host immune response using animal experimental system is required to avoid undesired outcome of experimental manipulation. Following severe influenza virus infection in influenza hemagglutinin antigen-specific transgenic mouse experimental model, step-wise evolving cells from a pool of naïve hemagglutinin-specific CD4+ T cells were studied for phenotypic, genomic, and functional characterization in vivo. Naïve CD4+ T cells respond with Th1 commitment in the absolute majority. They first develop into LAG-3Med IFN-γ-secreting Th1 effectors and then evolve into LAG-3High IFN-γ-not-secreting regulators with increasing LAG-3 expression upon continuous activation and cell division. The LAG-3Med IFN-γ-secreting effectors contribute to inflammation, boost inflammatory response of cognate antigen-specific CD8+ T cells, and aggravate the disease despite facilitated virus clearance. In contrast, LAG-3High regulators do not contribute to inflammation, suppress CD8+ T cell inflammatory response, alleviate lung pathology, and ameliorate the disease with preserved virus clearance. Moderated CD8+ T cells retain proliferative capacity, and persist beyond virus clearance. Such moderation is distinct from Foxp-3+ regulator-mediated suppression, which suppresses proliferative and inflammatory responses of the CD8+ T cells and impairs virus clearance with inflammation alleviation. Origin of regulatory from the effector cells of LAG-3-marked Th1 immunity alleviates lung inflammation without impairment of virus eradication.

Occurrence of Shiga toxin-producing Escherichia coli carrying antimicrobial resistance genes in sheep on smallholdings in Bangladesh.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are zoonotic foodborne pathogens and a significant concern with the emergence of antibiotic resistance. Close human contact might have a higher chance of being transmitted to humans from sheep if the sheep population is a potential reservoir of antimicrobial-resistant STEC. Therefore, this study aimed to examine the sheep population in rural Bangladesh for antimicrobial-resistant STEC. METHODS: We screened 200 faecal samples collected from sheep in three Upazilas from the Chattogram district. Randomisation of sampling was not performed due to the smaller flock size (two to six animals per smallholding). Phenotypically positive E. coli isolates were examined for two Shiga toxin-producing genes - stx1 and stx2. PCR-positive STEC isolates were investigated for the presence of antimicrobial resistance (AMR) genes - blaTEM , sul1 and sul2. RESULTS: In total, 123 of the 200 tested samples were confirmed positive E. coli using culture-based methods. PCR results show 17 (13.8%) E. coli isolates harboured ≥ one virulent gene (stx1 or/and stx2) of STEC. The AMR profile of STEC isolates was determined utilising the disc diffusion method. Of the STEC isolates, 82, 76, 71 and 71% were susceptible to chloramphenicol, gentamicin, ciprofloxacin and ampicillin. In contrast, 47% of isolates were resistant to trimethoprim-sulfamethoxazole, and 41% were resistant to amoxicillin. In addition, six of the tested STEC isolates exhibited the blaTEM  gene; eight STEC isolates had the sul1 gene, and the sul2 gene was detected in ten STEC isolates. CONCLUSION: To our knowledge, this study is the first to reveal a substantial percentage of STEC isolated from sheep in rural Bangladesh harbouring AMR genes.

Prevalence of coagulase-positive methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in dogs in Bangladesh.

BACKGROUND: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have a significant health impact on people with direct or supportive occupations in veterinary medicine including veterinarians, animal handlers, laboratory personnel and pet owners. OBJECTIVES: This cross-sectional survey was conducted to determine the prevalence of and risk factors for S. aureus, S. pseudintermedius, MRSA and MRSP in dogs in Bangladesh. METHODS: A total of 358 swab samples were collected from different body sites of 150 dogs attending a university teaching veterinary hospital between January and June 2018. Standard bacteriological procedures were followed to isolate Staphylococcus, and identification was confirmed to the species level by PCR to detect the nuc gene. MRSA and MRSP were confirmed by the presence of the mecA gene. RESULTS: The prevalence of coagulase-positive S. aureus and S. pseudintermedius in dogs were 16% and 45.3%, respectively. S. aureus and S. pseudintermedius isolates displayed the highest resistance against nalidixic acid (95.2% and 91%, respectively) and erythromycin (89.3% and 84.7%, respectively). Notably, all the staphylococcal isolates showed resistance to ≥3 antimicrobial classes. The prevalence of MRSA and MRSP in dogs was 8.7% and 6%, respectively. Multivariable logistic regression analysis identified the following variables as risk factors for MRSA colonisation in dogs: dogs with dermatitis (odds ratio [OR], 12.24, 95% CI: 3.12-57.33; p < 0.001) and history of antibiotic use (OR 8.73, 95% CI: 2.23-43.10; p < 0.001). Presence of otitis (OR 14.22; 95% CI: 1.64-103.58; p = 0.008) and oral lesions (OR 9.48, 95% CI: 1.14-64.82; p = 0.002) were identified as the significant risk factors for the carriage of MRSP. CONCLUSIONS: The circulation of multidrug-resistant S. aureus and S. pseudintermedius is a serious concern to dogs and humans. To our knowledge, this is the first report of S. pseudintermedius and MRSP affecting dogs in Bangladesh.

Antimicrobial resistance profiling and burden of resistance genes in zoonotic Salmonella isolated from broiler chicken.

BACKGROUND: Salmonella is frequently found in poultry of which only motile serovars have zoonotic significance due to their potential to induce human gastrointestinal infections. Antimicrobial resistance, being a public health concern, the emergence of multidrug-resistant (MDR) Salmonella serotypes affecting food chain has greater impact worldwide. AIM: Information on circulation of zoonotic Salmonella strains in commercial poultry farm level is limited in many parts of the world. This cross-sectional study was aimed to investigate the zoonotic Salmonella strains circulating in the broiler farm environment with their detailed antimicrobial resistance profiling. METHODS: Pooled faecal samples were collected randomly from commercial broiler farms of Chattogram district, Bangladesh. Standard bacteriological procedure was followed to isolate Salmonella, and identification was confirmed by genus specific polymerase chain reaction (PCR). After phenotypic characterisation of resistance profile against eight antimicrobials by disc diffusion technique, all strains were screened by PCR for some selected resistance genes. RESULTS: Out of the 350 samples, Salmonella was isolated and identified from 86 samples. In antimicrobial sensitivity testing, more than 98.8% isolates showed resistance to ampicillin and 94.2% to tetracycline followed by enrofloxacin (56%) and ciprofloxacin (50%). Notably, 94% isolates were found to be MDR. The results of PCR assays revealed that 81.4% of the isolates were carrying the tetA gene, 19.8% the tetB and 10.47% the tetC gene. The prevalence of the isolates bearing the blaTEM , blaCTX-M and Sul-I gene were 95.4%, 7.0 % and 37.2 %, respectively. CONCLUSION: There is a great risk to secure healthy poultry products due to the circulation of these MDR zoonotic Salmonella.

Relative COVID-19 Viral Persistence and Antibody Kinetics.

A total of 15 RT-PCR confirmed COVID-19 patients were admitted to our hospital during the in-itial outbreak in Taiwan. The average time of virus clearance was delayed in seven patients, 24.14 ± 4.33 days compared to 10.25 ± 0.56 days post-symptom onset (PSO) in the other eight pa-tients. There was strong antibody response in patients with viral persistence at the pharynx, with peak values of serum antibody 677.2 ± 217.8 vs. 76.70 ± 32.11 in patients with delayed versus rapid virus clearance. The patients with delayed viral clearance had excessive antibodies of compromised quality in an early stage with the delay in peak virus neutralization efficacy, 34.14 ± 7.15 versus 12.50 ± 2.35 days PSO in patients with rapid virus clearance. Weak antibody re-sponse of patients with rapid viral clearance was also effective, with substantial and comparable neutralization efficacy, 35.70 ± 8.78 versus 41.37 ± 11.49 of patients with delayed virus clearance. Human Cytokine 48-Plex Screening of the serial sera samples revealed elevated concentrations of proinflammatory cytokines and chemokines in a deceased patient with delayed virus clear-ance and severe disease. The levels were comparatively less in the other two patients who suf-fered from severe disease but eventually survived.

Role of Pneumococcal NanC in the Severe Disease of Streptococcus pneumoniae Superinfection with Influenza.

Bacterial superinfection aggravates the disease of influenza. Streptococcus pneumoniae is the most common bacterial pathogen. Synergistic virulence has been demonstrated between influenza neuraminidase and pneumococcal NanA and NanB. NanC, the other pneumococcal neuraminidase infrequently present in clinical isolates, is not well characterized. In this study, we report that superinfection with a NanC-negative pneumococcus strain suppresses anti-influenza immunity and impairs viral clearance with higher TGF-ß activation in mice. Bacterial load in the lungs also increases as the host immunity is suppressed. NanC-positive isogenic mutant reverses wild type S. pneumoniae-mediated immune suppression and facilitates virus clearance. However, it causes more severe disease as the augmented inflammation causes collateral damage. Both virus-mediated damage and immune response-mediated inflammation are important for pathogenesis of severe influenza. Inflammation may be more critical than virus-mediated damage in influenza with bacterial superinfection.

Abundance of Mobilized Colistin Resistance Gene (mcr-1) in Commensal Escherichia coli from Diverse Sources.

Aims: Antimicrobial resistance (AMR) spreads not only by pathogenic but also by commensal bacteria, and the latter can become a reservoir for resistance genes. This study was aimed to investigate the AMR patterns along with the presence of mobilized colistin resistance (mcr) genes in commensal Escherichia coli circulating in chickens, farm environments, street foods, and human patients. Materials and Methods: By a cross-sectional survey, isolates obtained from 530 samples were tested for their AMR profiles against 9 antimicrobials. Minimum inhibitory concentration (MIC) of the phenotypically colistin-resistant isolates was determined and screened for a set of mcr genes followed by sequencing of mcr-1 gene in the multidrug-resistant (MDR) isolates. Results: A total of 313 E. coli strains were isolated and confirmed by polymerase chain reaction. Antimicrobial susceptibility testing revealed that about 98% (confidence interval [95% CI] 95-99) of the isolates were MDR, and 58% (95% CI 52-63) isolates exhibited resistance to colistin. MIC values of colistin against the isolates ranged from 4 to 64 mg/L. Except for human patients, 20.4% colistin-resistant isolates from other sources of isolation had mcr-1 gene. Conclusions: There is abundance of commensal MDR E. coli strains with the acquisition of mcr-1 gene circulating in chickens and farm environments in Bangladesh.

Polymorphism of fecundity genes (BMP15 and GDF9) and their association with litter size in Bangladeshi prolific Black Bengal goat.

Goat farming in Bangladesh is primarily centred on indigenous Black Bengal goat, a highly prolific breed. Searching for genetic markers associated with prolificacy in this breed is vital for the country's goat breeding industry. However, there are no reports on polymorphisms associated with the fertility of Bangladeshi Black Bengal goats. This study investigated two major fecundity genes-bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to detect any possible mutations in these two genes associated with litter size in Black Bengal goats. Blood samples were collected from 40 raised goats in Hathazari Government Goat Farm, Bangladesh. Genomic DNA was extracted; PCR amplification was performed; and sequencing of PCR products was performed to detect polymorphism loci in the target genes. Five SNPs viz. C735A, C743A, G754T, C781A and C808G were detected in exon 2 of BMP15 gene. A SNP (T1173A) was detected in GDF9 exon 2. Association results show that SNPs at the 735, 754 and 781 nucleotide positions of BMP15 exon 2 had a significant association with litter size in Black Bengal goat. The effect of parity was also highly significant (P < 0.001) on litter size. For the first time, this study explored SNP loci in fecundity genes in Bangladeshi prolific Black Bengal goats. Further studies with many genetically unrelated animals for assessing the association of these loci and others in the fecundity genes with litter size may be useful.

Prevalence and antimicrobial susceptibility profiles of non-typhoidal Salmonella isolated from chickens in Rajshahi, Bangladesh.

Salmonellosis in poultry is an important disease that seriously impedes the development of the poultry industry. The increased resistance to antimicrobials against Salmonella has been a major public health concern worldwide. We conducted a study from January to June 2016 in and around the Rajshahi district of Bangladesh on the commercial chicken to isolate, identify and characterize poultry-specific Salmonella, to assess the potential risk factors and to determine the antimicrobial resistance pattern of the isolates. The overall prevalence of Salmonella enterica was 41% (49/120) [95% CI: 31.95%-50.17%] with 41.7% in broiler chicken (25/60) [95% CI: 29.06%-55.12%] and 40% in layer chicken (24/60, 40%) [95% CI: 27.56%-53.46%]. Samples collected from Rajshahi city (OR = 1.37, 95% CI: 0.50-3.73) and Puthia Upazila (OR = 1.51, 95% CI: 0.56-4.12) were more likely to be positive for Salmonella than Charghat Upazila. Salmonella detection was 1.3 times higher in chicken, providing loose feed than those provided ready feed. All the isolates fermented dextrose, maltose and mannitol with the production of acid and gas, but did not ferment sucrose and lactose. The isolates showed catalase, MR, citrate utilization test and TSI agar test positive, but indole and V-P tests negative. Salmonella isolates were sensitive to ciprofloxacin (90%), gentamycin (80%), amoxicillin (75%), streptomycin (70%), ampicillin (45%) and sulfamethoxazole-trimethoprim (45%), whereas highly resistant to penicillin (100%) and nalidixic acid (100%) followed by sulfamethoxazole-trimethoprim (55%), ampicillin (40%) and amoxicillin (25%). Salmonella enterica is endemic in commercial chicken production in Bangladesh with high prevalence. A considerable proportion of Salmonella isolates was found to be resistant to the majority of the common antimicrobial drugs. A good biosecurity system could be effective for the reduction of Salmonella. It is necessary to obtain universal commitments to establish prudent antibiotic use policies.

Circulation of oxytetracycline- and ciprofloxacin-resistant commensal Escherichia coli strains in broiler chickens and farm environments, Bangladesh.

BACKGROUND AND AIM: The emergence of antimicrobial resistance (AMR) in commensal organism, such as Escherichia coli of food animals, is an alarming issue for global health. It increases the possibility of transmitting AMR determinant(s) to human bacterial pathogens by transferable genetic materials, particularly by plasmids. Hence, it is important to know which resistant genes are being carried by commensal organisms in food chain in a country and their level of temporal loads. As a result, pre-emptive measures can be advocated with an aim to reduce their risks in their primary source of circulation which consequently would benefit the public health. MATERIALS AND METHODS: Commensal E. coli strains from broiler chickens on randomly selected 30 farms and the farm environments were examined for the frequencies of isolation of resistant strains to oxytetracycline and ciprofloxacin. Five birds were randomly selected from each farm to collect cloacal swab samples (total of 150 samples). Furthermore, a total of 150 environmental samples comprising one each from feed, water, soil, litter, and litter damping site of each farm were screened for the isolation of commensal E. coli strains. Strains thus obtained were initially tested for their resistance to oxytetracycline and ciprofloxacin by Kirby-Bauer disk diffusion method. Oxytetracycline-resistant strains were further screened for the presence of resistance determining genes, namely, tetA, tetB, and tetC by uniplex polymerase chain reactions. Risks associated with the isolation frequency of oxytetracycline- and ciprofloxacin-resistant E. coli were also assessed by univariable logistic regression analysis. RESULTS: The results revealed that all E. coli isolates, regardless of the source of origin, were resistant to oxytetracycline, while 78.4% (95% confidence interval [CI] 69.1-85.5%) showed resistance to ciprofloxacin. All the randomly selected (20) oxytetracycline-resistant strains harbored the tetA gene, whereas tetB and tetC were reported in three and two isolates, respectively. After univariable analysis, only one variable, that is, strain 1 of broiler chickens compared to two other strains was found to be positively associated with the isolation of ciprofloxacin-resistant E. coli (odds ratio 12.75 [95% CI 1.0-157.1], p=0.047). CONCLUSION: Resistance emerged against oxytetracycline and ciprofloxacin in commensal E. coli strains circulating in live poultry and farm environments in Bangladesh seems to be very high. Thus, human infection with drug-resistant E. coli strains through food chain will critically compromise the therapeutic measures currently available.

Coagulase-positive methicillin-resistant Staphylococcus aureus circulating in clinical mastitic goats in Bangladesh.

BACKGROUND AND AIM: Staphylococcus aureus is argued as one of the principal organisms responsible for mammary gland infection in lactating goats, causing both clinical and subclinical mastitis. Being highly zoonotic potential, pathogen emergence of methicillin-resistant S. aureus (MRSA) has a significant clinical impact on treatment and management of clinical mastitis. We conducted a cross-sectional study to investigate the prevalence of coagulase-positive S. aureus (CoPS), antimicrobial resistance profile of Staphylococcus spp., prevalence of MRSA, and association between different clinical parameters with CoPS. MATERIALS AND METHODS: A total of 67 clinical mastitic goats were sampled based on clinical examination and California mastitis test. Standard bacteriological methods were performed to isolate and identify Staphylococcus spp. CoPS were confirmed by nuc gene using polymerase chain reaction (PCR). All staphylococcal isolates were further examined for antimicrobial susceptibility testing by disk diffusion method. MRSA was confirmed based on mecA gene-based PCR. RESULTS: Here, 49 (73.13%; 95% confidence interval [CI], 61.41-82.35) samples were positive for Staphylococcus spp., of which 17 (34.69%; 95% CI, 22.88-48.73) isolates were CoPS and rest of the isolates (32; 65.30%; 95% CI, 51.27-77.12) were identified as coagulase-negative Staphylococcus spp. (coagulase-negative staphylococci [CNS]). Both, CoPS and CNS isolates displayed the highest resistance against tetracycline (76.47% and 75%, respectively) and oxacillin (70.58% and 68.75%, respectively). Notably, all staphylococcal isolates were multidrug-resistant (showed resistance to ≥3 classes of antimicrobials). mecA gene was found in 6 (8.96%; 95% CI, 3.84-18.52) CoPS isolates indicating MRSA strains. Among different clinical parameters, presence of high body temperature (p<0.05), firm udder consistency (p<0.01), bloodstained milk (p<0.00), and pus in milk (p<0.00) were significantly associated with the presence of CoPS in mastitic caprine milk. CONCLUSION: To the best of our knowledge, this is the first report of MRSA isolated from clinical caprine mastitis cases in Bangladesh. The findings of this study would help in cautious selection as well as administration of antimicrobials for therapeutic management of mastitic goats.

Acquisition of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli of Livestock Origin in Bangladesh.

Aims: To investigate plasmid-borne colistin resistance mechanism (plasmid-mediated colistin resistance [mcr-1]) in Escherichia coli of human, veterinary, and environmental origin in Bangladesh. Materials and methods: A total of 810 samples were collected from different sources. Isolation and identification of E. coli was performed using classical bacteriology and then tested for antimicrobial susceptibility. Colistin-resistant isolates were further analyzed for mcr-1 gene using PCR. Minimum inhibitory concentration (MIC) was determined using microbroth dilution technique. After sequencing of mcr-1 gene, phylogenetics was conducted to see the relationship with other mcr-1 gene sequences. Results: A total of 358 E. coli were isolated from 810 samples of humans, animals, environment, and food in Bangladesh. Of them 49 (15.9%) isolates were phenotypically resistant to colistin and 254 (70.9%) were resistant to multiple antimicrobials. mcr-1 gene was detected in three E. coli isolates of poultry source. For the three mcr-1 positive isolates the MIC of colistin sulfate was 4, 8, and 128 µg/mL. Gene sequencing of two of the three mcr-1 positive isolates and phylogenetic analysis showed close similarities of one isolate to other mcr-1 sequences available at GenBank while the other appeared to have evolved locally. Conclusion: First-ever report on circulation of mcr-1 E. coli of livestock origin in Bangladesh.

Effect of aloin on viral neuraminidase and hemagglutinin-specific T cell immunity in acute influenza.

BACKGROUND: Millions of people are infected by the influenza virus worldwide every year. Current selections of anti-influenza agents are limited and their effectiveness and drug resistance are still of concern. PURPOSE: Investigation on in vitro and in vivo effect of aloin from Aloe vera leaves against influenza virus infection. METHODS: In vitro antiviral property of aloin was measured by plaque reduction assay in which MDCK cells were infected with oseltamivir-sensitive A(H1N1)pdm09, oseltamivir-resistant A(H1N1)pdm09, H1N1 or H3N2 influenza A or with influenza B viruses in the presence of aloin. In vivo activity was tested in H1N1 influenza virus infected mice. Aloin-mediated inhibition of influenza neuraminidase activity was tested by MUNANA assay. Aloin treatment-mediated modulation of anti-influenza immunity was tested by the study of hemagglutinin-specific T cells in vivo. RESULTS: Aloin significantly reduced in vitro infection by all the tested strains of influenza viruses, including oseltamivir-resistant A(H1N1)pdm09 influenza viruses, with an average IC50 value 91.83 ± 18.97 µM. In H1N1 influenza virus infected mice, aloin treatment (intraperitoneal, once daily for 5 days) reduced virus load in the lungs and attenuated body weight loss and mortality. Adjuvant aloin treatment also improved the outcome with delayed oseltamivir treatment. Aloin inhibited viral neuraminidase and impeded neuraminidase-mediated TGF-ß activation. Viral neuraminidase mediated immune suppression with TGF-ß was constrained and influenza hemagglutinin-specific T cell immunity was increased. There was more infiltration of hemagglutinin-specific CD4+ and CD8+ T cells in the lungs and their production of effector cytokines IFN-γ and TNF-α was boosted. CONCLUSION: Aloin from Aloe vera leaves is a potent anti-influenza compound that inhibits viral neuraminidase activity, even of the oseltamivir-resistant influenza virus. With suppression of this virus machinery, aloin boosts host immunity with augmented hemagglutinin-specific T cell response to the infection. In addition, in the context of compromised benefit with delayed oseltamivir treatment, adjuvant aloin treatment ameliorates the disease and improves survival. Taken together, aloin has the potential to be further evaluated for clinical applications in human influenza.

Antibiotic resistant zoonotic bacteria in Irrawaddy squirrel (Callosciurus pygerythrus).

Irrawaddy squirrel (Callosciurus pygerythrus) may play an important role in the transmission of zoonotic bacteria, but little is known about the carriage of zoonotic bacteria in this common frugivorous rodent in Bangladesh. We aimed to investigate the presence of common zoonotic bacterial pathogens in Irrawaddy squirrel in the southeast part of Bangladesh. A total of 27 rectal and 27 oro-nasal swabs were collected from 27 healthy wild Irrawaddy squirrels. Four common zoonotic bacteria were isolated following routine laboratory procedures, and were identified based on colony morphology, and biochemical and staining properties. The pathogenic potential of the identified bacteria was confirmed by detection of virulence genes by PCR. All isolates were subjected to antimicrobial susceptibility test against seven antibiotics from six generic groups which are commonly used in human and veterinary medicine in Bangladesh. The prevalence of Escherichia coli, Salmonella spp., Yersinia spp. and Staphylococcus spp. was 44.4% (95% CI, 32.0-57.6), 13% (95% CI, 6.1-24.7), 44.4% (95% CI, 32.0-57.6), and 72.2% (95% CI, 59.0-82.5), respectively. We identified potential zoonotic virulence genes in all of these four bacterial species. Antimicrobial susceptibility testing revealed the presence of several multidrug resistant bacterial strains in squirrels. To the best of our knowledge, this is the first report in Bangladesh of the detection of antibiotic resistant zoonotic bacteria in Irrawaddy squirrels. The findings underpin the role of Irrawaddy squirrel as a source of pathogenic antibiotic resistant bacteria, consequently, fruit rejected because of squirrel consumption and squirrel-bites deserve more concern than previously.