Nontyphoidal Salmonella: a potential anticancer agent.

Use of bacteria in cancer therapy, despite being considered as a potent strategy, has not really picked up the way other methods of cancer therapies have evolved. However, in recent years, the interest on use of bacteria to kill cancer cells has renewed considerably. The standard and widely followed strategies of cancer treatment often fail either due to the complexity of tumour biology or because of the accompanying side effects. In contrast, these limitations can be easily overcome in a bacteria-mediated approach. Salmonella is a bacterium, which is known for its ability to colonize solid or semisolid tumours more efficiently than any other bacteria. Among more than 2500 serovars of Salmonella, S. Typhimurium has been widely studied for its antagonistic effects on cancer cells. Here in, we review the current status of the preclinical and the clinical studies with a focus on the mechanisms that attribute the anticancer properties to nontyphoidal Salmonella.

Whole-genome-based analysis reveals multiclone Serratia marcescens outbreaks in a non-Neonatal Intensive Care Unit setting in a tertiary care hospital in India.

We report the use of next generation sequencing (NGS) for investigating an outbreak of 13 cases of Serratia marcescens blood stream infections in a non-Neonatal Intensive Care Unit (non-NICU) setting in a tertiary care hospital in India over 5 months. Thirteen cases of sepsis due to S. marcescens were identified in various Intensive Care Units (ICUs) over 5 months. Environmental surveillance identified isolates in the adult ICU (AICU). Antibiogram did not correlate with timeline. Sequencing libraries were prepared using Nextera XT chemistry (Illumina). Based on NGS, two clusters were identified. Cluster 1 had environmental and clinical isolates from the AICU and cluster 2 were isolates from the Coronary Care Unit (CCU). NICU and Paediatric ICU isolates did not belong to any cluster. Polyclonal outbreaks best identified by NGS can occur simultaneously. Good infection prevention practices like hand hygiene for compounded medicines and surface cleaning helped end the outbreak.

T4-like Escherichia coli phages from the environment carry blaCTX-M.

The resistance determinant blaCTX-M has many variants and has been the most commonly reported gene in clinical isolates of extended spectrum beta-lactamase producing Escherichia coli. Phages have been speculated as potential reservoirs of resistance genes and efficient vehicles for horizontal gene transfer. The objective of the study was to determine the prevalence and characterize bacteriophages that harbour the resistance determinant blaCTX-M . Escherichia coli specific bacteriophages were isolated from 15 samples including soil and water across Mangaluru, India using bacterial hosts that were sensitive to ß-lactams. Phenotypic and genotypic characterization based on plaque morphology, host range, restriction fragment length polymorphism (RFLP), presence of blaCTX-M and electron microscopy was performed. Of 36 phages isolated, seven were positive for Group 1 of blaCTX-M . Based on host range and RFLP pattern, the seven phages were classified into four distinct groups, each harbouring a variant of blaCTX-M . Five phages were T4-like Myoviridae by electron microscopy which was further confirmed by polymerase chain reaction (PCR) for T4 specific gp14. Generalized transduction of the CTX-M gene from these phages was also observed. The high prevalence (20%) of this gene blaCTX-M in the phage pool confirms the significant role of Myoviridae members, specifically T4-like phages in the dissemination of this resistance gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The CTX-M gene that confers resistance to Beta-lactam class of drugs is widespread and diverse. Understanding mechanisms of antimicrobial resistance transfer is a key to devise methods for controlling it. Few studies indicate that bacteriophages are involved in the transfer of this gene but the type of phages involved and the degree of involvement remains to be explored. Our work has been able to identify the class of phages and the magnitude of involvement in the dissemination of this gene.

Differential expression of virulence genes and role of gyrA mutations in quinolone resistant and susceptible strains of Salmonella Weltevreden and Newport isolated from seafood.

AIMS: To investigate the differential expression of virulence genes and role of gyrA mutations in quinolone resistant and susceptible strains of Salmonella isolated from seafood. METHODS AND RESULTS: Forty Salmonella isolates from seafood were tested for antibiotic sensitivity. Minimal inhibitory concentration (MIC) was determined and two nalidixic acid-resistant isolates, viz Salmonella Weltevreden (SW9) and Salmonella Newport (SN36) were selected for identifying the mechanism of resistance. SW9 showed mutation in the gyrA gene at codon 83 (Ser to Tyr) while SN36 presented at codon 87 (Asp to Asn). Experimental induction of resistance to a sensitive Salm. Newport (SN71) showed point mutation at codon 87 (Asp to Gly) in the gyrA gene, and was designated SN71R. All the isolates resistant to nalidixic acid had a single mutation at different positions in the gyrA gene. However, induction of resistance to a sensitive Salm. Weltevreden (SW30) was exceptional in that it did not show any mutation in the gyrA region. Use of Phe-Arg-ß-naphthylamide (PAßN) also could not reduce MIC below the Clinical and Laboratory Standards Institute guidelines revealing the absence of efflux mediated resistance. Thus, the resistance mechanism in SW30R is unknown. The growth rate of quinolone resistant isolates was slower than the susceptible ones. The resistant isolates showed decreased epithelial cell invasion and intracellular replication. The mRNA expression levels of some of the genes were significantly (P < 0·005) reduced in SN71R compared to the sensitive strain (SN71). CONCLUSIONS: Nalidixic acid-resistant Salmonella strains are associated with lower virulence and pathogenicity than the sensitive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided valuable information on the difference in the growth, cytotoxicity, infectivity and expression of virulence genes in resistant and susceptible strains. Furthermore, the gyrA mutation was shown to be the main mechanism of quinolone resistance in Salmonella other than the overexpression of efflux pumps or the presence of plasmid mediated quinolone resistance genes.

Prevalence of adhesive genes among uropathogenic Escherichia coli strains isolated from patients with urinary tract infection in Mangalore.

The study was carried out to detect the adhesive genes pap (pyelonephritis associated pili), sfa (S fimbrial adhesin) and afa (afimbrial adhesin) from Escherichia coli strains isolated in patients diagnosed with urinary tract infection (UTI). A total of 23% of the isolates were positive for pap, sfa and afa genes with a prevalence of 60.87% (14/23), 39.1% (9/23) and 39.1% (9/23), respectively. Prevalence of multiple adhesive genes was 8.7% (2/23) for pap and afa, 30.43% (7/23) for pap and sfa. Significant numbers of isolates were positive for Congo red binding (80%) and haemolysin production 60%. The prevalence of multiple adhesive genes indicate the potential to adhere and subsequently cause a systemic infection among UTI patients.

Presence of T3SS2ß genes in trh⁺ Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India.

UNLABELLED: Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2α and T3SS2ß gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SSß genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2ß genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2ß genes are solely associated with trh⁺ and tdh⁺ /trh⁺ strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2ß genes identified in trh⁺ V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2ß-positive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: T3SSs (α or ß) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2ß genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2ß genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2ß genes in trh⁺ V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for seafood consumers.

Detection of class 1 integrons in Salmonella Weltevreden and silent antibiotic resistance genes in some seafood-associated nontyphoidal isolates of Salmonella in south-west coast of India.

AIMS: To study the antibiogram of 40 seafood isolates of Salmonella and use of PCR to detect the presence of integrons and genes coding for antibiotic resistance. METHODS AND RESULTS: In this study, 40 isolates of Salmonella were used for antibiogram analysis. The multidrug-resistant isolates were analyzed for the presence of integron using integron-specific primers. Twenty-five percentage of the isolates were multidrug resistant while 67·50% were resistant to at least two antibiotics. Antibiotic resistance genes catA1 and tetA were present in 57·52 and 60%, respectively. Although widespread presence of genes was observed, only 26·08% of the catA1-carrying isolates exhibited phenotypic resistance against the respective antibiotic. Integrons present in representative isolates of Salmonella Weltevreden and Salmonella Newport were sequenced. The former contained class 1 integron with a single gene dfrA7 in the integron cassette and an adjacent dihydropteroate synthetase gene along with the usual quaternary ammonium compound resistance gene, while the later contained class 1 integron with dhfrA1, OrfC, in the integron cassette and an adjacent dihydropteroate synthetase gene along with the usual quaternary ammonium compound resistance gene. CONCLUSIONS: This study demonstrates the presence of silent antibiotic resistance genes and class I integrons in seafood-associated Salmonella strains. The study also demonstrates the first report of class I integron in Salm. Weltevreden. Detection of catA1 genes in phenotypically sensitive bacteria suggests that these could be reservoirs in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The manuscript provides novel results describing the existence of a high rate of antibiotic resistance in the Salmonella populations prevailing in environmental sources as well as an absence of correspondence between the presence of antibiotic resistance genes, and the exhibition of a the corresponding phenotypic trait of resistance against the respective antibiotic compound was observed. In addition, the manuscript reports the presence of the class I integron in Salm. Weltevreden.

Monodon baculovirus of shrimp.

Among the viruses infecting penaeid shrimp, monodon-type baculovirus (MBV) otherwise known as Penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV), is one of the widely reported and well described viruses. It is a rod-shaped, enveloped, double-stranded DNA virus, and considered till recently, as the type A baculovirus. Besides MBV, two strains of SNPV are reported-plebejus baculovirus and bennettae baculovirus. MBV was reported to be originated from Taiwan and has wide geographic distribution and is reported to be enzootic in wild penaeids of the Indo-pacific coasts of Asia. The virus also has diverse host-range including a variety of cultured and captured shrimp species and freshwater prawn, Macrobrachium rosenbergii. MBV has been reported in all life stages of P. monodon with late larval, postlarval and young juvenile as the most susceptible stages/ages. However, MBV has not been documented in early larval stages. Although MBV has been reported to be tolerated well by shrimp, the infection has been attributed to decreased productivity. The target organs or tissues of MBV are the hepatopancreatic tubules and duct epithelium of postlarvae, juveniles and adults, and the anterior midgut epithelium of very young postlarvae. The prominent clinical sign of infection is the presence of multiple spherical inclusion bodies in the hepatopancreas and midgut epithelial cells. The major mode of transmission of the virus is horizontal through oral exposure to occlusion bodies, contaminated tissues or fomites. Minor morphometric variation of the virus has been reported among different isolates. The rod-shaped enveloped virus particles range from 265-324 nm in length and 42-77 nm in diameter. Although complete genome sequence of MBV is not available, nucleic acid of MBV is circular, double-stranded DNA with a genome size ranging from 80 to 160 kbp. The virus codes for a 53 kDa major polyhedrin polypeptide and two minor 47 and 49 kDa polypeptides. A variety of diagnostic tools have been reported for this virus including real-time PCR and LAMP-based detection. Taxonomic position is still uncertain and International Committee on Taxonomy of Viruses lists MBV as a tentative species named PemoNPV in the genus Nucleopolyhedrovirus. However, according to the latest genomic information on the virus, it has been suggested to create a new group of non-occluded bacilliform viruses called nudiviruses with MBV as one of the members. The aim of the current work is to describe the knowledge on the status, distribution and host-range, pathology, transmission, virus structure and morphogenesis, genomic characteristics, diagnosis and the latest taxonomic position of MBV.

Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (Artemia franciscana).

AIMS: This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii. METHOD AND RESULTS: Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts. CONCLUSION: Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation.

Presence of typical and atypical virulence genes in vibrio isolates belonging to the Harveyi clade.

AIMS: The study was aimed at investigating the presence of typical and atypical virulence genes in isolates belonging to the Harveyi clade (Vibrio harveyi and Vibrio campbellii). METHODS AND RESULTS: Forty-eight vibrio isolates belonging to the Harveyi clade were screened for the presence of virulence genes that are typical for these bacteria and those found in human pathogenic vibrios such as Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus and aquatic pathogenic Vibrio anguillarum. The virulence genes were amplified by PCR with specific primers, and the presence further confirmed by dot blot hybridization. The virulence genes vhh, chiA, vhpA, toxR(Vh), luxR and serine protease, typical of Harveyi clade were detected in all the isolates. The haemolysin gene hlyA and the virulence regulator gene toxR(Vc) specific to V. cholerae and the V. anguillarum-specific flagellum gene (flaC) were present in some of the isolates. Challenge tests with gnotobiotic Artemia nauplii did not show any correlation between the presence of the virulence genes and virulence of the isolates. CONCLUSION: From our results, there appears a remote possibility that vibrios belonging to the Harveyi clade might acquire virulence genes from other vibrios in the aquatic environment through horizontal gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios belonging to the Harveyi clade may be an important reservoir of virulence genes of other (human pathogenic) Vibrio species in the aquatic environment. The acquisition of virulence genes by horizontal transfer might increase the ability of Harveyi clade vibrios to infect aquatic organisms by increasing their virulence to a specific host by broadening their host range. The detection of such genes may forewarn the hatchery operators about a potentially virulent pathogen and thus help to develop management measures to handle the problem of vibriosis.

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

AIMS: The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp. METHODS AND RESULTS: The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli. Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila. Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations. CONCLUSIONS: The ompW-based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila. Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas. Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli, Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus, V. harveyi, V. alginolyticus, V. vulnificus. Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India.

AIMS: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR. METHODS AND RESULTS: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0.95, while ERIC-PCR showed a DI of 0.94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. CONCLUSIONS: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC-PCR generates useful information in the case of V. parahaemolyticus associated with seafood. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the usefulness of nucleic acid and protein-based studies in understanding the relationship between various isolates from seafood.

Contamination of community water sources by potentially pathogenic vibrios following sea water inundation.

Potentially pathogenic members of the Vibrionaceae family including Vibrio cholerae and Vibrio parahemolyticus were isolated from domestic sources of drinking water in coastal villages following sea water inundation during the tsunami in Southern India. Phenotypic and genotypic studies were done to confirm the identity and detection of toxins. Vibrio-gyr (gyrase B gene) was detected in all sixteen vibrio isolates. Toxin regulating genes i.e.: ctx gene, tdh gene, and trh gene, however were not detected in any of the strains, thereby ruling out presence of toxins which could endanger human life. Other potentially pathogenic bacteria Aeromonas and Plesiomonas were also isolated from hand pumps and wells, in a few localities. There was no immediate danger in the form of an outbreak or sporadic gastroenteritis at the time of the study. Timely chlorination and restoration of potable water supply to the flood affected population by governmental and nongovernmental agencies averted waterborne gastroenteritis. Assessment of quality of water and detection of potential virulent organisms is an important public health activity following natural disasters. This work highlights the importance of screening water sources for potentially pathogenic microorganisms after natural disasters to avert outbreaks of gastroenteritis and other infectious diseases.

Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India.

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.