Serological evidence of HIV, Hepatitis B, C, and E viruses among liver disease patients attending tertiary hospitals in Osun State, Nigeria.

Hepatitis infection in HIV positive individuals with liver diseases causes high mortality worldwide. HIV worsens the pathological effect of hepatitis viruses and potentiates reactivation of latent hepatitis infections due to reduced immunity. This research therefore aimed to study the occurrence of HIV and hepatitis viruses among liver diseases patients (LVDP) attending tertiary hospitals in Osun State, southwestern Nigeria. A total of 121 LVDP blood samples collected were tested for HIV and Hepatitis B, C, and E using and enzyme linked Immunossorbent assay (ELISA). Data were analyzed using packages within SPSS and P ≤ 0.05 was considered significant. Prevalence of 32.2%, 0.8%, 10.7%, and 18.2% for HBsAg, Anti-HCV, HEV-IgM, and HIV were found respectively. Marital status showed a significant association with HEV-IgM infection (χ2 = 9.869, P = .020). The prevalence of HBsAg, HEV, and HIV among LVDP in Osun State is alarming and health education among the patients and general populace is hereby advocated. High HEV-IgM seroprevalence implies that HEV routine screening should be incorporated into blood screening. Since HEV is associated with unhygienic practice, people should be enlightened on how to improve their living conditions.

High seroprevalence of viral hepatitis among animal handlers in Abeokuta, Ogun State, Nigeria.

Viral hepatitis is a deadly disease which can manifest as acute, chronic, hepatocellular carcinoma, and liver failure. Information about hepatitis is scarce among animal handlers. Due to Federal Government of Nigeria diversification programmes, many people are now involved in animal farming which can make them susceptible to viral hepatitis. This study aimed at determining the prevalence of Hepatitis B, C, and E viruses among animal handlers in Abeokuta, southwestern Nigerian. A total of 156 subjects were recruited for the study. Sociodemographic and risks factors data were fetched from subjects using interviewer-administered questionnaire. Blood samples were collected via venepuncture and tested for HCV, HBV, and HEV using ELISA technique. Results were analyzed using SPSS software version 21.0 and P value ≤ 0.05 was considered significant. The prevalence of HCV, HBV, and HEV were 46 (29.5%), 20 (12.8%), and 4 (2.6%) respectively while 6 (3.8%), 1 (0.6%), and 1 (0.6%) had co-infection of HBV-HCV, HBV-HEV, and HCV- HEV respectively. This study concludes that there is high prevalence of hepatitis C and B viruses among animal handlers in Abeokuta, Ogun state which is of significant public health problem, warranting further attention and research.

High Viral Hepatitis Infection among Pregnant Women Attending Antenatal Clinic in Adeoyo Maternity Teaching Hospital Ibadan (AMTHI) Oyo State, Nigeria.

Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Hepatitis E Virus (HEV) are highly endemic in several African countries including Nigeria with adverse effects on pregnancy outcomes resulting in fatality. This study aimed to determine the viral hepatitis in pregnant women attending antenatal clinic, AMTHI. Informed consent questionnaire was administered before blood collection via venipuncture. a total of 904 pregnant women plasma samples were tested for HBV, HCV, and HEV using ELISA kit. Data was analyzed using packages within SPSS software and P ≤ 0.05 was considered significant. Out of 904 samples analyzed, the overall prevalence of hepatitis infections among pregnant women attending antenatal clinic in AMTHI was 66(7.3%). High prevalence of the hepatitis infections was found among young women within the age group 21-30 which might be associated with active sex, intravenous drug use, sharing of sharp objects and alcoholism. Blood group O Positive had the highest prevalence of hepatitis. There was statistical significance between blood group and HBsAg infection (P < .05). Genotype AA women had highest prevalence of hepatitis. This study showed significant association between HBsAg, HCV, and HEV positive status with blood group O positive and Genotype AA pregnant women.

CTX-M-15 is Established in Most Multidrug-Resistant Uropathogenic Enterobacteriaceae and Pseudomonaceae from Hospitals in Nigeria.

ß-Lactam antibiotics are widely used to treat urinary tract infections in Nigeria. This study aimed to determine the presence and characteristics of extended spectrum ß-lactamases in commonly isolated uropathogenic Gram-negative bacteria (GNB) in Nigeria. Fifty non-duplicate GNB isolates consisting of Escherichia coli, 19; Klebsiella pneumoniae, 21; and Pseudomonas aeruginosa, 10 were obtained from three tertiary hospitals in Nigeria. The antibiotic susceptibility testing of all isolates to a panel of antibiotics including minimum inhibitory concentrations (MICs) and extended spectrum ß-lactamases was determined. Polymerase chain reactions and sequencing were used to detect ß-lactam genes. Polymerase chain reactions and sequencing identified varying extended spectrum ß-lactamases (ESBLs) encoding genes for 24 isolates (48.0%). Cefotaximase-Munich (CTX-M) 15 was the dominant gene with 20/24 of the isolates positive at 83.3%; multiple genes (2 to 6 ESBL genes) were found in 20 of the isolates. The isolates encoded other genes such as CTX-M-14, 33.3%; sulfhydryl variable (SHV) variants, 58.3%; oxacillinase (OXA) variants, 70.8%; OXA-10, 29.2%; and Vietnamese extended ß-lactamase (VEB) 1, 25.0%. There was no difference between the MIC50 and MIC90 of all the isolates. The high-level multidrug resistance of uropathogens to third generation cephalosporins including other antibiotics used in this study is strongly associated with carriage of ESBLs, predominantly CTX-M-15, as well as CTX-X-M-14, OXA-10, and VEB-1.

Knowledge and willingness of young adult to participate in early HIV vaccine trial in Nigeria

Nigeria has the second largest HIV epidemic (3.4 million) in the world with 3.2% of her young adults infected. Knowledge and willingness of young adults to participate in early HIV vaccine trial (EHVT) are essential for future interventions. This study aimed to investigate factors influencing willingness to participate (WTP) in EHVT. A cross-sectional study was employed to fetch data from 750 young adults (18-40years) recruited by systematic random sampling between June to December 2016. An informed consent questionnaire addressing socio-demographic factors, contraceptive practices, risky behaviours, knowledge and perception of EHVT study was completed by the participants. Data were analyzed using SPSS version 20 software and p ≤ 0.05 considered significant. Up to 240 (32.0%) of 750 expressed WTP in a vaccine study. There was a significant association between the WTP with; education levels (P=0.001), knowledge about HIV vaccine trial (HVT) studies (P=0.003); a positive insight toward the study (P=0.001); and age group 18-20years (P=0.001). Unwillingness to participate was associated with concerns about fear of reverting back, side effect, fear of spouse, use of parenteral route for its administration. Up to 684 (91.2%) of 750 knew contraceptive was for childbirth control, 241 (32.1%) has never used contraceptive while 172 (23%) used it during last coitus. Refusal to use contraceptive was associated with: religion, its side effect, not married, spouse un-approval, and ignorance. There was a significant association between the WTP with: education level, knowledge about HIV vaccine trial (HVT); a positive insight toward the study; and age group 14-20 years

Comparison of E-test with other conventional susceptibility testing methods for ciprofloxacin and gentamicin against gram negative enteric bacilli.

BACKGROUND: Increasing antibiotic resistance in Gram negative bacteria has led to the need for a faster and reliable method for determining antimicrobial susceptibility testing. In a resource poor setting like ours, it's also important to look for methods that will be clinically and economically beneficial to the patient. AIM: This study was aimed at evaluating the Epsilometer test (E-test) and conventional methods for determining antimicrobial susceptibility of isolates of Gram-negative enteric bacteria to ciprofloxacin and gentamicin. METHODS: Disc diffusion, E-test, broth dilution and agar dilution methods were performed on 54 bacterial isolates. RESULTS: Using the E-test, 88.9% of bacterial isolates were resistant to ciprofloxacin, 92.6% were resistant using broth microdilution, 96.3% were resistant using agar dilution and 72.2% were resistant using disc diffusion. Minimum inhibitory concentration (MIC50) of isolates for gentamicin showed significant difference for all the techniques (p < 0.05) while MIC90 for gentamicin and MIC50 and MIC90 for ciprofloxacin for all the techniques had no significant difference (p > 0.05). Both E-test and broth dilution methods showed high levels of agreement (p > 0.05), there were low levels of agreement between E-test and agar dilution method (p < 0.05), especially at MIC50. CONCLUSION: The E-test can therefore be considered a reliable method to determine antimicrobial susceptibility testing and it gives results which are at least as accurate as those obtained by the broth dilution method.

The non-association of Panton-Valentine leukocidin and mecA genes in the genome of Staphylococcus aureus from hospitals in South Western Nigeria.

PURPOSE: Virulence genes play important roles in pathogenesis of infections caused by S. aureus. The aim of this study was to determine the prevalence of PVL, eta and mecA genes in S. aureus isolated from patients in South-Western Nigeria. MATERIALS AND METHODS: In this study, a total of 116 S. aureus isolates from the clinical specimens submitted to laboratories in tertiary hospitals in the South Western Nigeria were used. Antibiotic susceptibility test was carried out to determine the susceptibility pattern of the isolates using multiple antibiotics disc. Minimum inhibitory concentration (MIC) was also carried out to determine the degree of resistant of the isolates to methicillin. PCR was used to screen for the presence of PVL, eta, and mecAgenes. RESULTS: mecA gene was detected in 48 (41.4%) of 116 strains of S. aureus. The MIC 50 and MIC 90 for mecA negative strains were 1 and 8 µg/ml, respectively while the MIC 50 and MIC 90 for mecA positive were >256 µg/ml. Twenty eight (24.1%) of 116 isolates were PVL gene positive with none of them mecA+. The prevalence of community acquired MRSA (CA-MRSA) was estimated to be 6.9% using molecular techniques. No localization of mecA gene and PVL gene on the genome of the entire S. aureus strains studied. Site of isolation of organism /specimen type was found to be associated with the prevalence of PVL+ and mecA+ S. aureus (P< 0.01). CONCLUSION: This study concludes that the PVL+ MRSA is rare and the prevalence of CA-MRSA is low in South-Western, Nigeria.

Anti-leukemic and immunomodulatory effects of fungal metabolites of Pleurotus pulmonarius and Pleurotus ostreatus on benzene-induced leukemia in Wister rats.

BACKGROUND: The use of natural bioactive compounds in conventional chemotherapy is a new direction in cancer treatment that is gaining more research attention recently. Bioactive polysaccharides and polysaccharide-protein complexes from some fungi (edible mushrooms) have been identified as sources of effective and non-toxic antineoplastic agents. Selected oyster mushrooms (Pleurotus pulmonarius and P. ostreatus being local [Nigeria] and exotic strains, respectively) were cultured on a novel medium of yeast extract supplemented with an ethanolic extract of Annona senegalensis, and the antileukemic potential of their metabolites was studied. METHODS: Leukemia was successfully induced in Wister rats by intravenous injection (0.2 mL) of a benzene solution every 2 days for 3 consecutive weeks. The aqueous solution of fungal metabolites (20 mg/mL) produced by submerged fermentation was orally administered (0.2 mL) before, during, and after leukemia induction. Leukemia burden was assessed by comparing the hematological parameters at baseline and after leukemia induction. The immunomodulatory potential of the metabolites was assessed by using a phagocytic assay (carbon clearance method). The ability to enhance leukopoiesis was assessed by using the total leukocyte count. RESULTS: Leukemia induction resulted in significant anemia indices and leukocytosis (P<0.05) in the experimental rats. Both metabolites equally enhanced leukopoiesis and demonstrated phagocytic actions; P. ostreatus activity was significantly higher than that of P. pulmonarius (P<0.05). CONCLUSION: The metabolites exhibited profound antileukemic potential by suppressing leukemia and demonstrating immunotherapeutic activities on animals after oral administration in various experimental groups.

Anti-leukemic and immunomodulatory effects of fungal metabolites of Pleurotus pulmonarius and Pleurotus ostreatus on benzene-induced leukemia in Wister rats

BACKGROUND: The use of natural bioactive compounds in conventional chemotherapy is a new direction in cancer treatment that is gaining more research attention recently. Bioactive polysaccharides and polysaccharide-protein complexes from some fungi (edible mushrooms) have been identified as sources of effective and non-toxic antineoplastic agents. Selected oyster mushrooms (Pleurotus pulmonarius and P. ostreatus being local [Nigeria] and exotic strains, respectively) were cultured on a novel medium of yeast extract supplemented with an ethanolic extract of Annona senegalensis, and the antileukemic potential of their metabolites was studied. METHODS: Leukemia was successfully induced in Wister rats by intravenous injection (0.2 mL) of a benzene solution every 2 days for 3 consecutive weeks. The aqueous solution of fungal metabolites (20 mg/mL) produced by submerged fermentation was orally administered (0.2 mL) before, during, and after leukemia induction. Leukemia burden was assessed by comparing the hematological parameters at baseline and after leukemia induction. The immunomodulatory potential of the metabolites was assessed by using a phagocytic assay (carbon clearance method). The ability to enhance leukopoiesis was assessed by using the total leukocyte count. RESULTS: Leukemia induction resulted in significant anemia indices and leukocytosis (P<0.05) in the experimental rats. Both metabolites equally enhanced leukopoiesis and demonstrated phagocytic actions; P. ostreatus activity was significantly higher than that of P. pulmonarius (P<0.05). CONCLUSION: The metabolites exhibited profound antileukemic potential by suppressing leukemia and demonstrating immunotherapeutic activities on animals after oral administration in various experimental groups.

High-level parasitic contamination of soil sampled in Ibadan metropolis.

Soil transmitted helminthes infections are common chronic human infections worldwide, this has been recognized as an important health problem, particularly in developing countries. The study was conducted within Ibadan metropolis in Oyo State, south western Nigeria between September 2008 and March 2009 to determine the prevalence of intestinal parasite in soil samples within the city. A total of 102 soil samples were collected from different sources from five local government areas ranging from refuse dumps, vegetable farms, school play grounds, abattoir, hospital, vicinity of house, gutter and road side. Two different methods of concentrating ova/cysts of parasites were used to analyze the samples--the zinc sulphate floatation technique and concentrated glucose solution method. Fifty-seven (55.9%) soil samples were positive for one or more parasites. These included; hookworm (37.3%), Strongyloides stercoralis (20%), Entamoeba histolytica (18.7%), Ascaris lumbricoides (17.3%), Trichuris trichiura (6.7%) respectively. The total number of parasites recovered was 75 (73.5%) and 74 (98.7) of these were recovered by the zinc sulphate floatation technique while only 44% was recovered by the concentrated normal saline-glucose solution technique. This study thus established the high prevalence rate of intestinal parasites in the soil sampled in Ibadan city and this obviously is one major means by which residents are at risk of parasitic diseases and also one of the means of vegetable contamination.

The presence of intestinal parasites in selected vegetables from open markets in south western Nigeria.

Intestinal parasitic infections are among the most common infection worldwide. In recent years there has been an increase in the number of reported cases of food-borne illness linked to fresh vegetables which is a major way in the transmission of intestinal parasites. The study was carried out to determine the level of parasitological contamination of vegetables sold at selected markets in south western Nigeria. A total of 120 samples from different vegetables were randomly sampled from major selected open markets in 3 cities. The vegetables were analysed using macroscopic, sedimentation and magnesium sulphate floatation techniques. Eighty-two (68.3%) of the vegetables were positive for intestinal parasites from which water leaf (Talinium triangulare) and 'soko' (Celosis) recorded the highest (100%) parasitic contamination. Parasites detected were Ascaris lumbricoides (16.7%), hookworm (18.3%), Taenia spp (4.2%), Strongyloides stercoralis (45.8%), Balantidium coli (0.8%). Vegetables in each of these cities had almost the same high rate of parasitic contamination; Ibadan (70%), Ilorin (70%) and Lagos (65%). This study further emphasised the role of vegetables in the transmission of intestinal parasites in developing countries. Therefore, vegetable farmers should therefore be enlightened on the modern use of night soil as fertilizer and the treatment of irrigation water or municipal waste water before use. There is also dire need for the improvement of sanitary facilities in our markets and vegetable vendors should also be included in the screening of food handlers.

Development of infection model for studying intracellular gene expression of Mycobacterium tuberculosis.

Mycobacterium tuberculosis complex owe their ability to cause infection because of their intracellular survival ability in professional phagocytic cells of human and the ability to enter into stage ofdormancy. The aim of this study was to develop an infection model that could be used to study M. tuberculosis and macrophage interactions at molecular level. Four infection models were examined namely opsonised M. bovis BCG / J774.2 macrophage cell line, non-opsonised M. bovis BCG / J774.2 macrophage cell line, opsonised M. tuberculosis / J774.2 macrophage cell line, and non-opsonised M. tuberculosis / J774.2 macrophage cell line infection models. A J774.2 macrophage cell line was synchronously infected with M. bovis (BCG strain) and M. tuberculosis (H37Rv), respectively at different multiplicity of infections (M.O.I). For opsonisation, the organisms were pre-incubated with human serum prior to infection. The infected cell lines were examined by light microscopy and electron microscopy with viable bacterial counts. Macrophage viability was assessed by trypan blue exclusion staining. The results showed higher significant level of infection of J774.2 macrophage cell line by opsonised M. bovis BCG (30 - 40%) compared to non-opsonised M. bovis BCG (< 0.1%) at an M.O.I of 50 (p < 0.05) with high macrophage viability. In contrast, there was no significant statistical difference (p > 0.05) in high infectivity (30 - 42%) with high macrophage viability achieved with using non-opsonised M. tuberculosis and opsonised M. tuberculosis, respectively, at an M.O.I of 10. In conclusion, opsonisation is not required for M. tuberculosis / J774.2 infection model in contrast to M. bovis BCG / J774.2 infection model where opsonisation is necessary to achieve high level of infection.

The Use of Rap-Pcr in Studying Mycobacterium Tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes; which were specifically expressed; or upregulated during intracellular infection of J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA was differentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCRr2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway; a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected; none were specifically expressed during intracellular infection but bacilli

Asymptomatic bacteriuria of pregnancy in Ibadan, Nigeria: a re-assessment.

Asymptomatic bacteriuria in pregnancy is the major risk factor for developing symptomatic urinary tract infection during pregnancy. In the present study, 300 pregnant women are screened for significant asymptomatic bacteriuria in order to provide an insight into the prevalence in developing countries, reassessment of some predisposing factors and aetiological agents and their susceptibility tests. The mean age of the patients in the study is 26.8 years (SD: 5.8 years, range: 16-40 years). Using 10(3) organisms/mL as a significant level of bacteriuria, the prevalence was found to be 21.0%. One hundred and fifty-eight samples had no pus cells, with 25 showing significant bacteriuria, 116 samples contained 1-4 pus cells/high power field (hpf) with 25 showing significant bacteriuria, while 26 samples had > or = 5 pus cells/hpf with 13 showing significant bacteriuria. There was no particular trend associated with age and rate of infection. However, there was a decline in the rate of infection in the 26-30 age group, with a sharp increase as age increased. There was high incidence of bacteriuria during the third trimester of pregnancy (21.9%) compared with that in the first trimester (7.7%), while the level in the second trimester was 22.5%. Multiparity is associated with increased bacteriuria in pregnancy. Thirty-one (49.2%) isolates grew Gram-negative bacilli; 27 (42.9%) grew Gram-positive cocci and the remainder (7.9%) grew yeast-like cells. Staphylococcus aureus was the most frequent pathogen (41.3%), followed by Klebsiella species (33.3%) and Escherichia coli (11.1%). Bacterial isolates from this study were most sensitive to ceftazidime, followed by ceftriazone, and least susceptible to co-trimoxazole.

Comparative assessment of virulence traits in Legionella spp.

Legionella pneumophila is a facultative intracellular pathogen that accounts for the majority of cases of Legionnaires' disease in the USA and Europe, but other Legionella spp. have been shown to cause disease. In contrast, Legionella longbeachae is the leading cause of Legionnaires' disease in Australia. The hallmark of Legionnaires' disease caused by L. pneumophila is the intracellular replication within phagocytes in the alveolar spaces, and the Dot/Icm type IV secretion system is essential for intracellular replication. Although it has been presumed that intracellular replication within phagocytes is the hallmark of other virulent legionellae, the virulence traits of Legionella spp. apart from L. pneumophila are not well defined. In this study, 27 strains of Legionella spp. belonging to 16 species that have been isolated from humans or from the environment were examined for five virulence traits exhibited by L. pneumophila: cytopathogenicity, intracellular replication within macrophages, induction of apoptosis/DNA fragmentation, pore-formation-mediated cytolysis of the host cell, and the presence of the dot/icm loci. The strains were divided into two broad groups (low and high cytopathogenic groups) based on cytopathogenicity assays using U937 human-derived macrophages. The other four virulence traits were evaluated in the low and high cytopathogenic groups of Legionella species. Most L. pneumophila serogroup 1 strains were highly cytopathogenic after 72 h, manifested high levels of intracellular growth, induced apoptosis/DNA fragmentation, and exhibited pore-forming activity. The majority of the other species were the low cytopathogenic group that did not induce apoptosis, neither did they exhibit pore-forming activity. All the species of legionellae tested have all the dot/icm loci, when examined by DNA hybridization. No correlation was found between cytopathogenicity and the other four pathogenic traits.

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga.

We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DeltaG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation-mediated cytolysis and egress from mammalian cells. In contrast, the icmTDeltaG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore-forming activity. Consistent with their defect in pore formation-mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.

icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells.

The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila.

Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79-90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are "trapped" within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.