Coexistence of metallo-beta-lactamase-encoding genes in Pseudomonas aeruginosa.

BACKGROUND & OBJECTIVES: The emergence and rapid spread of carbapenem resistance mediated by metallo-beta-lactamase (MBL) in Pseudomonas aeruginosa is of major concern due to limited therapeutic options. This study was aimed at detecting the presence of MBL and its association with integrons in imipenem-resistant P. aeruginosa isolates and to determine their genetic relatedness. METHODS: A total of 213 P. aeruginosa isolates were collected from two tertiary care centres and tested against anti-pseudomonal antibiotics by antimicrobial susceptibility testing, followed by the detection of MBL production by combined disk method. Minimum inhibitory concentration (MIC) of meropenem was determined by E-test. Multiplex polymerase chain reaction (PCR) was performed for the detection of blaSPM, blaIMP, blaVIM, blaNDM, blaGIM and blaSIM. PCR was carried out to characterize the variable region of class 1 integron. Transcongujation assay was carried out for the confirmation of plasmid-mediated resistance. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR was performed for determining the genetic relatedness among P. aeruginosa isolates. RESULTS: Of the 213 P. aeruginosa isolates, 22 (10%) were found to be carbapenem resistant and these were from pus 18 (82%), urine 2 (9%), sputum 1 (5%) and tracheal wash 1 (5%). Among 22 isolates, 18 (81.8%) were found to be MBL producers by phenotypic method and MIC range of meropenem was 8 to >32 µg/ml. PCR amplification showed that 20 (91%) isolates carried any one of the MBL genes tested: blaVIM and blaNDM in seven (32%) and six (27%) isolates, respectively; blaVIM and blaNDMin three (14%); blaIMP and blaNDM in two (9%); blaVIM and blaIMP in one (5%) isolate. The blaVIM, blaIMP and blaNDM were found to co-exist in one isolate. None of the isolates were positive for blaSPM, blaSIM and blaGIM. All 22 isolates carried class I integron. Of the 20 MBL-positive isolates, transconjugants were obtained for 15 isolates. ERIC-PCR analysis showed all isolates to be clonally independent. INTERPRETATION & CONCLUSIONS: Our results showed 10.3 per cent of carbapenem resistance among P. aeruginosa isolates, and the coexistence of MBL-encoding genes among P. aeruginosa mediated by class I integron.

Viridans and bovis group streptococci that cause infective endocarditis in two regions with contrasting epidemiology.

Viridans group (VGS) or bovis group streptococci (BGS) are the major causes for streptococcal infective endocarditis (IE). However, the causative isolates are not sufficiently characterized. Using multilocus sequence analysis we have examined VGS and BGS (VGS/BGS) isolates that caused IE in southern India and Germany, two distant geographic regions with a contrasting IE epidemiology. Other than in Germany, the majority of patients (68%) in Chennai, southern India had an underlying rheumatic heart disease (RHD). In accord with the high prevalence of RHD in the younger population and with the expansive age structure of India, the median age (24 years) of the VGS/BGS endocarditis patients was lower than in Germany (63 years), where RHD is rare and the age structure is contractive. Both in Germany and in southern India, the majority of cases were caused by mitis group streptococci, however, with considerable differences in the spectra of causative (sub)species. BGS endocarditis was more frequent in Germany. The spectrum of VGS/BGS that cause IE differs considerably between distant geographic regions in which different predisposing conditions prevail. Therefore, improved microbiological diagnosis in IE may facilitate determination of the optimal therapy.

Low vaccine efficacy of mumps component among MMR vaccine recipients in Chennai, India.

Introduction of MMR vaccine was believed to have resulted in a decline in the incidence of measles, mumps and rubella infections. However, recent reports suggest the re-emergence of mumps infection worldwide in the vaccinated populations. It was proposed that the reason for this re-emergence was poor efficacy of MMR vaccine. The present study was aimed to investigate mumps infection in MMR vaccinated and non-vaccinated populations in Chennai, India. Blood samples were collected from acute mumps cases (n=74, 42<12 yr age, 54% males) and investigated for IgM antibody against mumps, IgG antibody against measles, mumps and rubella viruses by ELISA. Sixty seven (91%) patients had received MMR vaccine. All the 67 vaccinated cases were positive for parotitis, and mumps IgM. However, only 10 (15%) were positive for IgG. All samples (100%) were positive for rubella and measles IgG. These findings showed the occurrence of mumps infection among MMR vaccinated individuals in Chennai, India. The MMR vaccine failed to generate anti-mumps IgG. The reason may be low vaccine efficacy of the mumps component of the MMR vaccine used.

Uncommon pathogens causing infective endocarditis.

Infective endocarditis is caused by a wide range of aetiological agents. The microbiology, epidemiology, and treatment of this disease have changed considerably in the last two decades. Staphylococci and streptococci are known to be the classical causative agents; however, blood culture-negative endocarditis caused by fastidious and slow-growing organisms is now common. The list of uncommon pathogens causing endocarditis has expanded in recent years. This is a narrative literature review of the aetiological agents of endocarditis that are rarely encountered in clinical practice, their epidemiology, the characteristics of these pathogens, the clinical presentations of the cases, and their management.

16S rRNA sequencing as a diagnostic tool in the identification of culture-negative endocarditis in surgically treated patients.

BACKGROUND AND AIM OF THE STUDY: Infective endocarditis (IE) is a worldwide problem, and at least one-third of cases are culture-negative despite the use of appropriate laboratory techniques. METHODS: A broad-range polymerase chain reaction (PCR) amplification was performed of the 16S rRNA gene, followed by single-strand sequencing for 26 surgically removed heart valves from patients with culture-negative endocarditis who had undergone valve repair or replacement. RESULTS: Two of the 26 patients were PCR-positive, and sequencing of the amplicon identified the etiological agent. Gram-stained smears of the heart valves were positive in both cases. Three of the remaining 24 cases which were negative by PCR also showed the presence of micro-organisms in Gram-stained smears. CONCLUSION: The study results emphasize that, in suspected IE cases when there is no growth in culture, a combination of microscopy and 16S rRNA sequencing can be used to identify the pathogen in excised valvular tissue.

Genotyping of erythromycin resistant group C & G streptococci isolated in Chennai, south India.

BACKGROUND & OBJECTIVES: Increasing resistance to erythromycin has been observed worldwide in group C and group G streptococci (GCS/GGS). The information available from India is scanty. The aim of the study was to identify erythromycin resistant GCS/GGS isolates in Chennai, south India, and to compare erythromycin resistant genotypes with emm types. METHODS: One hundred and thirty one GCS/GGS isolates were tested for erythromycin resistance by disc diffusion and agar dilution methods. Erythromycin resistance genotypes [erm(A), erm(B) and mef(A)] were determined by a multiplex PCR. emm types of erythromycin resistant GCS/GGS isolates was also assessed using emm gene sequencing method. RESULTS: Sixteen of the 131 isolates (12.21%) were resistant to erythromycin. Majority of the isolates were GGS (15/16). Eight of the 16 (50%) were S. dysgalactiae subsps. equisimilis. Twelve isolates (75%) were MLS B phenotype and four (25%) were M phenotype. Of the 12 isolates which exhibited MLS B resistance, seven showed cMLS B phenotype and were positive for erm(B) gene. The remaining five were iMLS B phenotype of which three were positive for erm(A) gene and two for erm(B) gene. erm(A) was common among carriers whereas erm(B) was common among clinical isolates. INTERPRETATION & CONCLUSIONS: MLS B was the predominant phenotype and erm(B) was the common genotype in the present study. The emm type stC1400.0 was frequently associated with erythromycin resistant GCS/GGS in our study.

The oral microbiota in patients undergoing coronary artery bypass graft surgery.

The oral cavity houses a diverse community of microorganisms which play an important role in maintaining the health of the individual. The coexistence of periodontal infections with coronary artery disease (CAD) has been shown in many studies. We investigated the presence and abundance of periodontitis-causing bacteria in patients with CAD. The oral microbiome of five patients admitted for coronary artery bypass graft (CABG) surgery was analysed by metagenomic sequencing of 16 s ribosomal ribonucleic acid (rRNA) gene amplicons. Two samples of oral mouthwash were collected 48-72 h apart when the patients were in the intensive care unit. Abundance and diversity of oral bacterial flora were analysed. The most abundant phyla were Firmicutes and Bacteroidetes. Less than 5% of the taxa in this group of patients belonged to the phylum Proteobacteria. Though there were variations in the abundance of the bacterial species in the immediate postoperative period, there was no major change in the overall diversity. High counts of periodontal pathogens such as Tannerella forsythia, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, and Treponema denticola were seen in most of the patients.

Use of 16s rRNA Gene Sequencing for the Identification of Viridans Group Streptococci.

Streptococci belonging to the viridans group are gram-positive bacteria residing as commensals in the upper respiratory, gastrointestinal, and genital tracts in humans. Though they are largely known to be commensals, they may also cause life-threatening infections like infective endocarditis, septicemia, pyogenic infections, pneumonia, and meningitis. The viridans group streptococci (VGS) are usually identified by biotyping; however, species discrimination is not always possible by phenotypic characterization. We identified 53 isolates from blood cultures of patients with infective endocarditis and compared the results of biotyping with 16s rRNA gene sequencing for species identification. Organisms belonging to the mitis group were the most common. 16s rRNA gene polymerase chain reaction and sequencing were useful in identifying the etiological agents at the species level. S.oralis was the most common etiological agent.

Culture-negative endocarditis caused by Stenotrophomonas maltophilia: a report of two cases.

Stenotrophomonas maltophilia, an aerobic, non-fermenting, Gram-negative bacterium, is an emerging nosocomial pathogen. It is considered to be a low-grade pathogen, and infections due to S. maltophilia are uncommon. However, in the recent past, S. maltophilia infections have been on the rise, particularly in patients who are either immunocompromised, aged or on long-term antibiotic therapy. Endocarditis due to S. maltophilia is extremely rare. This is a report of two patients with S. maltophilia endocarditis who were diagnosed by 16S rRNA gene sequencing.

Molecular investigation of human metapneumovirus in children with acute respiratory infections in Chennai, South India, from 2016-2018.

Human metapneumovirus (hMPV) has emerged as a frequent cause of acute respiratory infections (ARI) among young children. The prevalence and genetic diversity of hMPV circulating in Chennai, Southern India, has not been studied yet. Hence, this study was aimed to investigate the prevalence, co-infection with other respiratory viruses like HRSV A and B, influenza A and B, hRV and HPIV 1-4 viruses, socio-demographic associations, and genotypes of hMPV among children in Chennai. A total of 350 nasal swab specimens were collected from children with ARI during April 2016 to August 2018 and tested for hMPV by real time PCR method. In this study, hMPV was detected in 4% (14/350) of the samples. One hMPV positive sample was found to be co-infected with influenza B virus. The mean and median ages of the children with hMPV infection were 61.5 months (5.1 years) and 83 months (6.9 years), respectively. Phylogenetic analysis of the partial F gene revealed the presence of A2c subcluster among the study strains as well as with B1 and B2 lineages. The prevalence data obtained in this study is important in evaluating the role of hMPV in childhood ARI and emphasizes the importance of routine viral diagnosis in hospitals. To the best of our knowledge, this is the first study to report the prevalence, seasonality, and genetic diversity of hMPV in Chennai as well as the first study to report A2c subcluster of hMPV among children in India.

Q fever endocarditis in India: A report of two cases.

Q fever is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii. Infective endocarditis is the most common form of chronic Q fever. Diagnosis of Q fever is difficult, as there are no pathognomonic symptoms. Methods of isolation of the organism in culture are tedious, hence serological and molecular techniques remain the mainstay of diagnosis. We report two cases of Q fever endocarditis diagnosed by IFA and real-time PCR.

Oral candidiasis caused by Kodamaea ohmeri in a HIV patient in Chennai, India.

Kodamaea ohmeri was isolated from a 38-year-old HIV seropositive woman with pseudomembranous oral candidiasis. The isolate was identified by the API 20 C yeast identification system and confirmed by sequence analysis. Antifungal susceptibility testing done by E-test showed that the isolate was susceptible to voriconazole, amphotericin B and caspofungin.

Environmental isolation of Cryptococcus neoformans and Cryptococcus gattii from living trees in Guindy National Park, Chennai, South India.

We report for the first time the environmental isolation of Cryptococcus neoformans from decaying wood and bark debris of living trees in Guindy National Park, Chennai, South India. Of the 40 trees screened, four isolates of Cryptococcus species were recovered of which two were Cryptococcus gattii, one was C. neoformans and one was untypable. The isolation of C. neoformans from Eucalyptus globulus and C. gattii from Cassia marginata in this study constitutes the first record of the natural occurrence of C. neoformans varieties in these tree species anywhere in the world. The isolation of C. gattii from Syzygium cumini represents the first isolation from South India.

Carriage of Candida species in oral cavities of HIV infected patients in South India.

Fifty-four patients with human immunodeficiency virus (HIV) infection were studied to assess the load of oral carriage of Candida spp. The mean oral Candida carriage density (30,305.93 +/- 56,643.93 CFU ml(-1)) in HIV patients was significantly higher than that seen in the control population (93.48 +/- 358.48 CFU ml(-1); P = 0.000). The mean Candida load in HIV patients with oral thrush (46,591.43 +/- 65,002.57 CFU ml(-1)) was significantly higher than in the HIV subjects without oral thrush (306.32 +/- 699.50 CFU ml(-1); P = 0.000). Non-C. albicans Candida species (56%) were more predominant than the C. albicans (44%) isolates. 25S rDNA PCR analysis of C. albicans revealed preponderance of genotype A strains. Interestingly, 42.6% of rinse specimens grew multiple Candida species, with the combination of C. albicans and C. krusei (39.1%) being the most frequent.

Genetic diversity of human respiratory syncytial virus in children with acute respiratory infections in Chennai, South India.

Introduction: Human respiratory syncytial virus (HRSV) an RNA virus belonging to Pneumoviridae family, is an important cause of acute respiratory infections (ARIs) in young children. HRSV circulates as two subgroups A and B, which are further categorised into several genotypes. New genotypes may replace existing ones over successive epidemic seasons and multiple genotypes may cocirculate in the same community rendering it important to monitor them at the molecular level. The present study assessed the circulating genotypes of HRSV in Chennai. Materials and Methods: Two hundred and sixty-seven children with ARI were recruited during the study from April 2016 to March 2018 for detecting HRSV A and B by real-time reverse transcription-polymerase chain reaction. Phylogeny and selection pressure analysis were done. Results: Fifty-seven of the 267 samples (21.3%) were positive for HRSV, of which 7.1% and 14.2% were HRSV A and B, respectively, indicating that HRSV B was the major subgroup circulating in Chennai. Peak activity of HRSV was observed during the monsoon and winter months. Phylogenetic analysis of 2nd hypervariable region (HVR) of attachment glycoprotein gene (G gene) revealed that the HRSV A strains belonged to ON1 and HRSV B strains belonged to BA9 genotypes. Several unique amino acid substitutions were observed among the study strains. The Shannon entropy plot revealed that the HRSV A strains from our study have a high potential for amino acid substitutions in the 2nd HVR of G gene. Conclusion: This study underlines the genetic diversity of HRSV and emphasises the need for continued molecular surveillance for infection management and prevention strategies.

Influenza Virus Among Children with Acute Respiratory Infections in Chennai, India.

Influenza is a major public health concern. Information on the prevalence of influenza virus in respiratory tract infections in Indian children is very sparse. In the present study, 267 nasal swabs were collected from children with acute respiratory infections in Chennai, India, out of which 22 (8.2%) and 6 (2.3%) samples were positive for influenza A and B virus respectively.

Comparison of nested polymerase chain reaction and real-time polymerase chain reaction targeting 47kda gene for the diagnosis of scrub typhus.

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October-February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.

Bactericidal activity of different types of honey against clinical and environmental isolates of Pseudomonas aeruginosa.

OBJECTIVES: Honey has had a valued place in traditional medicine for centuries. Renewed interest in honey for various therapeutic purposes, including treatment of infected wounds, has led to the search for different types of honey with antibacterial activity. In this study, we have assessed the antibacterial activity of different types of honey (manuka honey from Australia, heather honey from the United Kingdom, and locally marketed Indian honey). METHODS: The agar dilution method was used to assess the antibacterial activity of honey against 152 isolates of Pseudomonas aeruginosa by determining minimum inhibitory concentrations. RESULTS AND CONCLUSIONS: The locally available (khadikraft) honey produced the best activity against Pseudomonas aeruginosa and was found to be better than all of the imported varieties of therapeutic honey.