Detection of clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex and Klebsiella pneumoniae isolated from farmed freshwater fish.

AIMS: The present study was aimed to detect clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex (CR-ECC), Klebsiella pneumoniae (CR-KP), and Serratia plymuthica (CR-SP) isolated from farmed freshwater fish. METHODS AND RESULTS: Out of 243 spatially diverse freshwater fish samples analysed, 5.3% were contaminated with CR-ECC, 1.6% with CR-KP, and 0.4% with CR-SP. The CR-ECC was further identified as E. asburiae (38.5%), E. mori (23.1%), E. cloacae (15.4%), E. hormaechei (15.4%), and E. kobei (7.7%) by 16S rRNA gene sequencing. The CR-ECC were resistant to carbapenems and cefoxitin, whereas CR-KP and CR-SP were multi-drug resistant (MDR). The CR-ECC harboured the carbapenemase gene blaIMI alone or in combination with blaTEM, blaEBC, blaCIT, blaACC, and tet(E). Whereas, CR-KP harboured carbapenemase gene, blaNDM-5 along with blaOXA-48, blaSHV, blaOXA-1, blaCTX-M-15, tet(A), sul1, and qnrB. No carbapenemase-encoding genes were detected in CR-SP. The MLST analysis showed that CR-KP belonged to ST231 and ST1561 lineages, while CR-ECC did not show exact match with any reported STs. The plasmid replicons predominantly detected were IncF and IncI1. Broth mating assays of CR-KP and CR-ECC with recipient Escherichia coli J53 indicated that blaNDM-5 was transferable but not blaIMI. CONCLUSION: This study highlights the low-level contamination of carbapenem-resistant Enterobacterales (CRE) harbouring clinically relevant carbapenemase-encoding genes in farmed freshwater fish from India. The CR-ECC of fish origin did not show the potential to spread carbapenem resistance.

Molecular characterization of carbapenem resistant E. coli of fish origin reveals the dissemination of NDM-5 in freshwater aquaculture environment by the high risk clone ST167 and ST361.

Aquatic environment can act as reservoir and disseminator of antimicrobial resistance and resistant pathogens. Novel high-risk carbapenem resistant E. coli (CREC) are continuously emerging worldwide; however, the occurrence of CREC in freshwater aquaculture environment is largely unexplored. To fill this gap, large scale sampling of freshwater pond sites and retail fish markets was done between Oct 2020 and Oct 2021 to investigate the CREC contamination in fish. The frequency of CREC contamination in the freshwater fish was 6.99% (95% CI: 3.78-10.20%). All the isolates were MDR and harbored carbapenemase encoding gene, blaNDM-5 along with other antimicrobial resistance genes (ARGs), blaTEM (64.7%), blaCTX-M-15 (35.3%), blaOXA-1 (5.9%), tet(A) (100%), sul1 (94.1%), qnrS (82.3%), cat1 (35.3%), and cat2 (23.5%). The isolates belonged to phylogroup C and showed low virulence gene profile. ERIC-PCR grouped the isolates into five clusters (I-V). The isolates of clusters I, II, and III were identified as ST167 (76.4%) and of cluster IV as ST361 (17.6%). This is the first report documenting the contamination of NDM-5 producing E. coli ST167 and ST361 of clinical/livestock lineage in freshwater fish from India. The blaNDM-5 was significantly associated with ARGs, tet(A), and sul1; and plasmid replicons, IncF, IncI1, and IncP, signifying the presence of blaNDM-5 and associated ARGs on these transferable plasmids. These findings were validated by the successful conjugal transfer of blaNDM-5 and associated ARGs into non-CREC strain (J53). Our study highlights the ability of CREC to disseminate antimicrobial resistance which has health implications and environmental concerns.

Effectiveness of 13-valent pneumococcal conjugate vaccine on radiological primary end-point pneumonia among cases of severe community acquired pneumonia in children: A prospective multi-site hospital-based test-negative study in Northern India.

INTRODUCTION: Community acquired pneumonia (CAP) is a leading cause of under-five mortality in India and Streptococcus pneumoniae is the main bacterial pathogen for it. Pneumococcal Conjugate Vaccine 13 (PCV13) has been introduced in a phased manner, in the national immunization program of India since 2017/2018. The primary objective of this study was to evaluate the effectiveness of PCV13 on chest radiograph (CXR)-confirmed pneumonia, in children hospitalized with WHO-defined severe CAP. METHODS: This prospective, multi-site test-negative study was conducted in a hospital-network situated in three districts of Northern India where PCV13 had been introduced. Children aged 2-23 months, hospitalized with severe CAP and with interpretable CXR were included after parental consent. Clinical data was extracted from hospital records. CXRs were interpreted by a panel of three independent blinded trained radiologists. Exposure to PCV13 was defined as ≥2 doses of PCV13 in children aged ≤ 12 months and ≥ 1 dose(s) in children > 12 months of age. Our outcome measures were CXR finding of primary endpoint pneumonia with or without other infiltrates (PEP±OI); vaccine effectiveness (VE) and hospital mortality. RESULTS: From 1st June 2017-30th April 2021, among 2711 children included, 678 (25.0%) were exposed to PCV1. CXR positive for PEP±OI on CXR was found in 579 (21.4%), of which 103 (17.8%) were exposed to PCV. Adjusted odds ratio (AOR) for PEP±OI among the exposed group was 0.69 (95% CI, 0.54-0.89, p = 0.004). Adjusted VE was 31.0% (95% CI: 11.0-44.0) for PEP±OI. AOR for hospital mortality with PEP±OI was 2.65 (95% CI: 1.27-5.53, p = 0.01). CONCLUSION: In severe CAP, children exposed to PCV13 had significantly reduced odds of having PEP±OI. Since PEP±OI had increased odds of hospital mortality due to CAP, countrywide coverage with PCV13 is an essential priority.

Antibacterial activity of palmarosa oil significantly varies between Aeromonas veronii and Aeromonas caviae and exhibits selective action on tetracycline and sulfonamide resistant A. caviae.

AIMS: To investigate the antibacterial activity of three (palmarosa, basil and rosemary) essential oils (EOs) on Aeromonas veronii and Aeromonas caviae, and determine minimum inhibitory concentration (MIC) of potent EO against tetracycline and sulfonamide resistant strains. METHODS AND RESULTS: Palmarosa oil (PMO) showed significantly (p < 0.05) higher inhibition zones against both A. veronii and A. caviae (n = 30) than basil and rosemary in the disk diffusion assay. The MIC (% v/v) of PMO ranged from 0.008% to 1.00%. The mean MIC was significantly higher for A. caviae (0.48 ± 0.24%) than A. veronii (0.21 ± 0.15%). Further, the MIC of PMO was compared in six groups: Group 1: Tetracycline Resistant A. veronii (TRV); Group 2: Tetracycline Resistant A. caviae (TRC); Group 3: Sulfonamide Resistant A. veronii (SRV); Group 4: Sulfonamide Resistant A. caviae (SRC); Group 5: Susceptible A. veronii (SV) and Group 6: Susceptible A. caviae (SC). No significant differences were observed between overall resistant (TRV+ SRV) and susceptible A. veronii (SV). However, in A. caviae, the resistant group had a lower MIC than the susceptible group. Moreover, the MIC was significantly lower for TRC (0.31 ± 0.11%) as compared to SRC (0.46 ± 0.10%). The time of kill of PMO for both the species of Aeromonas was 20-30 min. CONCLUSION: Palmarosa oil exhibited significantly higher activity on A. veronii than A. caviae. The resistant strains of A. caviae were inhibited at a lower concentration than susceptible strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Palmarosa oil could be explored as an alternative antimicrobial agent for mitigating antimicrobial resistance and managing Aeromonas infection in fish and their risks to public health.

Effect of spatio-temporal variables, host fish species and on-farm biosecurity measures on the prevalence of potentially pathogenic Aeromonas species in freshwater fish farms.

AIMS: To determine the prevalence of Aeromonas species in freshwater fish farms, factors affecting their prevalence and virulence factors associated with each species. METHODS AND RESULTS: In a cross-sectional study from 128 farms in four districts of Uttar Pradesh, India, 11 species of Aeromonas were identified by gyrB sequencing including the first report of Aeromonas crassostreae from fish. Four species of Aeromonas were more prevalent (MP) in fish farms, A. veronii bv. sobria (50.0%) was the highest, followed by A. caviae (18.8%), A. veronii bv. veronii (11.7%) and A. dhakensis (7.0%). The less prevalent (LP) species were A. hydrophila, A. media, A. jandaei, A. allosaccharophila, A. salmonicida, A. crassostreae and A. taiwanensis. Spatial variation in the prevalence of Aeromonas species was observed. Dominance of biovar sobria ranged from 33.3 to 68.6%, notably lesser in farms with on-farm biosecurity measures. The prevalence of biovar veronii was significantly associated with pangas fish, rainy season and farms with on-farm biosecurity measures. The prevalence of LP species was significantly higher in mrigal fish and winter season. Multiple virulence factors (>6) were detected in 70.2% of the Aeromonas species. Significant association of ß-hemolysin, DNase, slime production, act, ahh1, aexT and lip was observed with different species of Aeromonas. Moreover, 75.8% of Aeromonas species possessed one or more enterotoxins genes (act/alt/ast). CONCLUSION: Significant association of spatio-temporal variables, host fish species and on-farm biosecurity measures were observed on the prevalence of some of the Aeromonas species in freshwater fish farms. Most of the Aeromonas species harboured virulence factors indicating their potential for pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that determined the prevalence and identified the factors that affect the prevalence of Aeromonas species in freshwater fish farms. This information will be useful in managing Aeromonas infection in fish and their risks to public health.

Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries.

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

Epidemiology of Hypoxic Community-Acquired Pneumonia in Children Under 5 Years of Age: An Observational Study in Northern India.

BACKGROUND: Community-acquired pneumonia (CAP) is the leading cause of under-five mortality in India. An increased risk of mortality has been reported in cases of hypoxic pneumonia. METHODS: The primary objective of this study was to assess the proportion of children aged 2-59 months, hospitalized with hypoxic CAP, as well as socio-demographic, clinical, and radiological features associated with it. The secondary objective was to determine the risk of mortality among hospitalized cases of hypoxic CAP. This prospective, observational study was conducted in four districts of Northern India, between January 2015 and April 2021. A hospital-based surveillance network was established. Inclusion criteria were as follows: (a) child between 2 and 59 months, (b) hospitalization with symptoms of WHO-defined CAP, (c) resident of project district, (d) illness of <14 days, and (e) child had neither been hospitalized for this illness nor recruited previously. Children whose chest x-rays (CXRs) were either unavailable/un-interpretable and those that received any dose of pneumococcal conjugate vaccine-13 were excluded. Hypoxic pneumonia was defined as oxygen saturation <90% on pulse oximetry or requiring oxygen supplementation during hospital stay. RESULTS: During the study period, 71.9% (7,196/10,006) children of severe pneumonia were eligible for inclusion, of whom 35.9% (2,580/7,196) were having hypoxic pneumonia. Female gender and use of biomass fuel for cooking increased the odds of hypoxic CAP. Clinical factors like wheezing, pallor, tachypnea, low pulse volume, presence of comorbidity, general danger signs, severe malnutrition, and radiological finding of primary end-point pneumonia ± other infiltrates (PEP±OI) also increased the odds of hypoxic CAP in a conditional logistic regression model. Adjusted odds ratio for mortality with hypoxia was 2.36 (95% CI: 1.42-3.92). CONCLUSION: Almost one-third of cases hospitalized with severe CAP had hypoxia, which increased chances of mortality. Besides known danger signs, certain newer clinical signs such as pallor and wheezing as well as PEP+OI were associated with hypoxic CAP. Therefore, objective assessment of oxygen saturation must be done by pulse oximetry in all cases of CAP at the time of diagnosis.

Effect of immunization of rohu Labeo rohita with inactivated germinated zoospores in providing protection against Aphanomyces invadans.

Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ±â€¯12 g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P < 0.05) protection (66.7% RPS). In addition, following booster immunization (before challenge), the antibody level in the GA group was significantly higher (P < 0.05) than the control group. In western blotting, sera from G and GA groups showed reactivity with peptides of about 54 KDa. Following challenge (on 14th day), the antibody level as well as total antiprotease activity in fish of all the groups was significantly decreased in comparison to pre-challenge, except fish in GA group not exhibiting any gross lesions. In addition, sera of surviving fish of GA group showed significant inhibition of germination of zoospores and germlings growth in comparison to other groups (P < 0.05). Further, histopathological examination of the muscle tissue revealed that, in fish of GA group without any gross lesions, there were well developed granulomas and extensive mononuclear cell infiltration restricted to the site of injection, whereas in other groups, there was extensive myonecrosis with proliferating hyphae. These preliminary findings indicate that inactivated germinated zoospores of A. invadans in combination with adjuvant could stimulate good immune response and confer remarkable protection in rohu.