The Utility of Maternal TORCH Screening Due to Obstetrical Indications in Detecting Congenital Infections: A Retrospective Observational Study.

BACKGROUND: The diagnostic yield of TORCH screening for obstetrical indications is unclear. We evaluated TORCH testing results among women with intrauterine growth restriction (IUGR), polyhydramnios and oligohydramnios; and associations with congenital infections in neonates. METHOD: This retrospective single-center study included all the women diagnosed with IUGR, polyhydramnios or oligohydramnios who underwent serological TORCH testing during 2010-2019. TORCH screening included Toxoplasma, cytomegalovirus (CMV), rubella IgM and IgG. The data, which were cross-referenced with data of neonates with congenital TORCH infections during the same period, included indications for neonatal testing, sonographic findings and neonatal ophthalmologic and hearing findings. RESULT: Six women of 771 (0.8%) were diagnosed with primary TORCH infection: 4 (0.5%) with toxoplasmosis, and 2 (0.3%) with CMV. None had a confirmed congenital infection. The rates of positive maternal TORCH screening in IUGR and polyhydramnios were 2.1% and 0.6%, respectively. Maternal TORCH infection was not identified in any woman with oligohydramnios or severe polyhydramnios. None of the neonates with congenital infection were screened for TORCH during pregnancy due to polyhydramnios, oligohydramnios or IUGR. Among the neonates with congenital CMV, the most common indication for performing neonatal CMV polymerase chain reaction was suspected primary maternal infection during pregnancy due to symptomatic CMV. No incidences of congenital rubella were noted in the last decade in our medical center. CONCLUSION: Our results suggest that routine TORCH screening in pregnancies complicated with IUGR, polyhydramnios or oligohydramnios should be avoided. Suggestive maternal symptoms and specific fetal sonographic features should prompt testing for CMV and Toxoplasma infection.

Antibiotic treatment of women with isolated intrapartum fever vs clinical chorioamnionitis: maternal and neonatal outcomes.

BACKGROUND: Clinical chorioamnionitis refers to the presence of maternal fever (≥38°C) and at least 2 clinical signs: (1) maternal tachycardia (>100 bpm), (2) fetal tachycardia (>160 bpm), (3) maternal leukocytosis >15,000/mm2, (4) purulent vaginal discharge, and (5) uterine tenderness. Few data exist to guide the appropriate management of women with isolated intrapartum fever in the absence of other clinical signs suggesting chorioamnionitis. OBJECTIVE: This study compared maternal and neonatal infectious outcomes and microbiological outcomes between women with isolated intrapartum fever and women with clinical chorioamnionitis. STUDY DESIGN: This 10-year retrospective study included all the laboring women at our institution, at ≥34 weeks of gestation, with a singleton pregnancy and body temperature of ≥38.0°C, with or without other evidences of infection. According to our department protocol, women with isolated intrapartum fever received intravenous ampicillin, whereas women with clinical chorioamnionitis received intravenous ampicillin plus gentamicin. The primary outcome was puerperal endometritis, compared between women with isolated intrapartum fever (treated with ampicillin) and women with clinical chorioamnionitis (treated with ampicillin plus gentamicin). The secondary maternal outcomes consisted of (1) maternal clinical outcomes, such as cesarean delivery, surgical site infection, postpartum hemorrhage, and postpartum length of stay, and (2) microbiological studies, including positive chorioamniotic membrane swabs and blood culture. Among the secondary neonatal outcomes were early-onset sepsis, neonatal intensive care unit admission, and length of stay. Of note, 2 multivariate logistic regression models were created. A model aimed to predict puerperal endometritis controlled for gestational age of >41 weeks, diabetes mellitus, obesity, positive group B streptococcus status, rupture of membrane ≥18 hours, meconium staining, positive chorioamniotic membrane swabs, cesarean delivery, and empiric postdelivery antibiotic administration. A model aimed to predict neonatal early-onset sepsis controlled for gestational age of 34 to 37 weeks, positive group B streptococcus status, rupture of membrane ≥18 hours, and positive chorioamniotic membrane swabs. RESULTS: Overall, 458 women met the inclusion criteria. Compared with women with clinical chorioamnionitis (n=231), women with isolated intrapartum fever (n=227) had higher rates of puerperal endometritis (3.9% vs 8.8%; P=.03), early-onset sepsis (0.4% vs 4.4%; P=.005), positive chorioamniotic membrane swabs (46.3% vs 63.9%; P<.001), and ampicillin-resistant Escherichia coli (35.5% vs 48.9%; P=.033). The rate of group B streptococcus-positive chorioamniotic membrane swabs was similar between the groups. In a subanalysis of women with negative or unknown group B streptococcus status, the puerperal endometritis and neonatal early-onset sepsis rates were higher among women with isolated intrapartum fever than women with suspected chorioamnionitis (8.7% vs 3.3% [P=.041] and 4.1% vs 0% [P<.001], respectively). In 2 multivariate analysis models, among women with isolated intrapartum fever treated with ampicillin compared with those with clinical chorioamnionitis treated with ampicillin and gentamicin, the odds ratio of antibiotic treatment of endometritis was 2.65 (95% confidence interval, 1.06-6.62; P=.036), and the odds ratio of neonatal early-onset sepsis was 8.33 (95% confidence interval, 1.04-60.60; P=.045). CONCLUSION: Women with intrapartum fever, with or without other signs of infection, were at increased risk of maternal and neonatal complications. The use of ampicillin as a sole agent in isolated intrapartum fever might promote ampicillin-resistant E coli growth in the chorioamniotic membranes and consequently lead to puerperal endometritis and early-onset sepsis. In this context, a broad-range antibiotic should be considered.

Time to reconsider antibiotic prophylaxis in women with prolonged rupture of membranes: The trend of Enterobacteriaceae in peripartum infections.

OBJECTIVE: The proportion of neonatal early-onset sepsis (EOS) by gram-negative bacteria has increased. The authors examined bacterial distribution in the amniotic membrane cultures of women with peripartum fever (PPF) and related perinatal outcomes. METHODS: This retrospective study covered the period 2011 to 2019. The primary outcomes were Enterobacteriaceae-positive birth culture rates in women with PPF and the trend of ampicillin resistance. Maternal and neonatal outcomes were compared between women with group B Streptococcus (GBS) and Enterobacteriaceae-positive isolates. Bacterial distribution was also compared according to rupture of membrane (ROM) duration. RESULTS: Among 621 women with PPF, the positive birth culture rate was 52%. Increasing prevalences of ampicillin-resistant Enterobacteriaceae (81%) were noted. Positive birth cultures were associated with maternal bacteremia (P = 0.017) and neonatal EOS (P = 0.003). Prolonged ROM ≥18 h was associated with increased risk for Enterobacteriaceae-positive cultures, while intrapartum ampicillin and gentamicin were associated with lower risk. Enterobacteriaceae-positive compared with GBS-positive birth cultures were associated with adverse maternal and neonatal outcomes. CONCLUSION: Positive birth cultures were related to maternal bacteremia and neonatal sepsis. Adverse outcomes were more prevalent among women with Enterobacteriaceae-positive versus GBS-positive birth cultures. Prolonged ROM is a risk factor for Enterobacteriaceae-positive birth cultures among women with PPF. Antibiotic prophylaxis treatment for prolonged ROM should be reconsidered.

Compared perinatal outcomes of two prophylactic antibiotic regimens for preterm premature rupture of membranes: a randomized controlled trial.

BACKGROUND: Prophylactic antibiotic use in preterm premature rupture of membranes is associated with significantly reduced intra-amniotic infection and improved neonatal outcome, although data are insufficient to determine the optimal antibiotic regimen. Ampicillin resistance has changed the epidemiology of neonatal sepsis. OBJECTIVE: This study aimed to determine the efficacy of two antibiotic regimens in prolonging the latency period in women with preterm premature rupture of membranes. STUDY DESIGN: This randomized-controlled trial was conducted in 3 tertiary university-affiliated hospitals. A total of 124 women with preterm premature rupture of membranes at <37 weeks of gestation were randomized into two antibiotic prophylactic protocols: ampicillin + roxithromycin and cefuroxime + roxithromycin. The latency period length, neonatal adverse outcomes, and maternal infectious morbidity, including intrauterine infection, intrapartum fever, postpartum antibiotic treatment, endometritis, and wound infection, were measured and compared. RESULTS: Maternal infectious morbidity was higher in the ampicillin group than in the cefuroxime group (17.7% vs 6.5%; 1-sided P value =.048). The pathogen distribution among placenta, membrane, cord, and uterine cultures differed between the groups (P=.017). Enterobacteriaceae spp. cultures were identified in 68.6% of the cultures in the ampicillin group and 43.2% in the cefuroxime group (P=.036). The composite neonatal adverse outcome was higher in the ampicillin group than in the cefuroxime group (55 [88.7%] vs 46 [74.2%]; 1-sided P value =.03). The proportion of primiparas with a latency period >4 days was significantly higher in the cefuroxime group than in the ampicillin group (odds ratio, 3.69; 95% confidence interval, 1.175-11.607; P=.025). CONCLUSION: In combination with roxithromycin, the use of cefuroxime, as a prophylactic in women with premature rupture of membranes at <37 weeks of gestation, showed longer pregnancy in primiparas and less maternal and neonatal morbidity than the use of ampicillin. Further larger studies are needed to support our results.

Maternal colonization with extended-spectrum ß-lactamase-producing Enterobacteriaceae in term versus preterm pregnancies.

OBJECTIVES: To examine the prevalence and risk factors of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) colonization among women who delivered preterm and at term. METHODS: A prospective observational study of maternal ESBL-E rectovaginal colonization in threatened preterm labor and low-risk term pregnancies was conducted between March 2017 and August 2021 at the Galilee Medical Center, Israel. Obstetric and neonatal complications were compared between colonized and non-colonized mothers and neonates. RESULTS: ESBL-E colonization was similar in the preterm (n = 202) and term (n = 172) groups: 14.4% and 16.9%, respectively (P = 0.567). The maternal-neonatal transmission rate was higher in the preterm than the term group but the difference was not statistically significant: 42.1% and 22.2%, respectively (P = 0.42). Prematurity was a risk factor of neonatal ESBL-E colonization (odds ratio 1.33, 95% confidence interval 1.01-1.75, P = 0.041). ESBL-E-colonized preterm infants were delivered at an earlier gestational age and were more likely to have complications. Maternal ESBL-E colonization and transmission were more prevalent in pregnancies complicated by threatened preterm labor or premature rupture of membranes than in term pregnancies. CONCLUSIONS: These findings emphasize the need for further research on the cost-effectiveness of screening for maternal ESBL-E colonization in preterm labor, to prevent neonatal infectious complications. CLINICALTRIALS: gov identifier NCT03251885.

Clinical implications of uterine cultures obtained during urgent caesarean section.

BACKGROUND: Postpartum endometritis (PPE) is 12-25 times more common following caesarean sections (CS) performed after labour onset than after vaginal delivery. Risk factors for PPE include prolonged rupture of membranes (ROM), chorioamnionitis, prolonged labour, multiple cervical examinations and Group B Streptococcus colonisation of the lower genital tract. AIMS: We compared uterine culture results and microbial antibiotic susceptibility according to ROM duration in emergent intrapartum CS. Secondary outcomes included PPE incidence, and identification of clinical and microbiological predictors of infectious postpartum morbidity. MATERIALS AND METHODS: In a retrospective case series of intrapartum CS in which uterine culture was performed, associations with postpartum outcomes including postpartum microbiology are reported. The results were stratified by the duration of ROM (treated as a categorical variable). Univariate analysis was performed. RESULTS: Positive uterine cultures were identified in 15% of emergent CS and correlated with prolonged ROM. Escherichia coli was the sole pathogen isolated in preterm CS; the ampicillin resistance rate was 75%. Among women with positive uterine cultures, rates were increased for postpartum fever, re-admission, PPE and surgical site infection. Cultures obtained from postpartum infections correlated with pathogens isolated from uterine cultures during CS in 46.1% of women. Positive uterine culture was related to umbilical cord pH < 7.1 (P = 0.017). CONCLUSIONS: Obtaining routine intrauterine culture during intrapartum CS is of low risk and low cost, and relatively easy to perform. Further research should investigate clinical and health economic impacts of obtaining intrauterine culture during CS, influences on postpartum antibiotic treatment, and maternal and neonatal morbidity.

Clearance of the SARS-CoV-2 Virus in an Immunocompromised Patient Mediated by Convalescent Plasma without B-Cell Recovery.

Coronavirus disease (COVID-19) is a contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This case report presents a patient who had difficulty eradicating the corona virus due to being treated with Rituximab, which depletes B lymphocyte cells and therefore disables the production of neutralizing antibodies. The combined use of external anti-viral agents like convalescent plasma, IVIG and Remdesivir successfully helped the patient's immune system to eradicate the virus without B-cell population recovery. In vitro studies showed that convalescent plasma is the main agent that helped in eradicating the virus.

Vertical Transmission of Extended-Spectrum, Beta-Lactamase-Producing Enterobacteriaceae during Preterm Delivery: A Prospective Study.

Maternal carriage and vertical transmission of extended-spectrum, beta-lactamase-producing Enterobacteriaceae (ESBL-E), such as Escherichia coli, hamper the treatment of infections, resulting in high morbidity. E. coli is the most frequent cause of early-onset neonatal sepsis (EOS) in preterm infants, where ESBL-E are more frequently isolated. In this prospective, case-controlled study, maternal rectovaginal ESBL-E colonization and vertical transmission to preterm infants were assessed in 160 women with preterm premature rupture of membranes (PPROM; 57.4%) or preterm labor (42.6%); additional cultures were obtained from the placenta, amnion, and umbilical cord during preterm labor. Maternal and neonatal ESBL-E-carriage rates were 17.5% and 12.9%, respectively, and the vertical-transmission rate was 50%. Maternal ESBL-E colonization among women with PPROM was 21.3%, and in women with premature labor it was 12.6%. No correlation was observed between maternal ESBL-E-colonization and previous hospitalization or antibiotic administration during pregnancy. However, a correlation was found between placental inflammation and maternal ESBL-E colonization (p = 0.007). ESBL-E-colonized infants were delivered at an earlier gestational age and were more likely to have complications. Thus, the high ESBL-E carriage rate in women with threatened preterm labor, without obvious risk factors for carriage, and a high vertical transmission rate, combined with a correlation between placental inflammation and ESBL-E carriage, support maternal-neonatal ESBL-E-colonization surveillance and active measures to prevent ESBL-E-related EOS.

A novel extended prophylactic antibiotic regimen in preterm pre-labor rupture of membranes: A randomized trial.

OBJECTIVES: Prophylactic antibiotic use in preterm pre-labor rupture of membranes (PPROM) is associated with a significant reduction in intra-amniotic infection and improved neonatal outcome. However, data is insufficient to determine the optimal antibiotic regimen. Considering the rise in Escherichia coli and Klebsiella pneumonia early-onset sepsis rate and the emergence of ampicillin resistance, our aim is to compare the efficiency of two antibiotic regimens in prolonging pregnancy and reducing infectious morbidity. DESIGN: This multicenter randomized unblinded controlled prospective trial compared two antibiotic prophylactic protocols in PPROM: ampicillin + roxithromycin vs. cefuroxime + roxithromycin in 84 women with PPROM, from 12/2015-12/2019. RESULTS: The median latency period was significantly longer (p = 0.039) in the cefuroxime + roxithromycin group (4.63 [0.59-50.18] days) than in the ampicillin + roxithromycin group (2.3 [0.15-58.3] days). Neonatal admission to neonatal intensive care unit rate, hospitalization length, neonatal respiratory distress syndrome, neonatal fever, and need for respiratory support or mechanical ventilation, were similar between the groups. K. pneumonia cultures were significantly more frequent in the ampicillin + roxithromycin group. None of the cultures were group B Streptococcus positive. CONCLUSIONS: To prolong latency period and reduce gram-negative early-onset sepsis, cefuroxime + roxithromycin is recommended as the first-line protocol in PPROM. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02819570.

Routine uterine culture swab during cesarean section and its clinical correlations: A retrospective comparative study.

OBJECTIVES: Cesarean sections, particularly non-elective cesareans, are an important risk factor for the development of postpartum endometritis, a leading cause of postpartum febrile morbidity. We evaluated the yield of obtaining routine intrauterine culture during elective and non-elective cesarean sections, in the prevention and management of postpartum endometritis. STUDY DESIGN: A retrospective comparative study investigating the distribution of uterine cultures obtained immediately after fetus and placenta delivery during cesarean sections performed in a single tertiary hospital during 2017. True pathogenic bacteria were included in the study analysis and considered as positive results, while other contaminant bacteria were excluded. RESULTS: Positive uterine cultures were identified in 10.7 % (88/821) of cesarean sections, with no significant difference in prevalence between elective and non-elective cesareans. Escherichia coli (E.coli), isolated in 40.9 % of the positive cultures of all women, was the most common organism in non-elective cesareans vs. Group B Streptococcus (GBS) in elective cesareans. Higher rate of positive cultures was found in term vs. preterm cesareans (17.5 % vs 10.5 %, respectively, p-value = 0.04). E.coli was the most frequent pathogen reported in both women with intact membranes or premature rupture of membranes (46.3 % and 47.3 % respectively). Eight women (9.1 %) with positive cultures presented with postpartum fever; all had undergone non-elective cesarean section. In one-third of these cases the empirical antibiotic treatment was adjusted according to the uterine culture results and susceptibility testing results. CONCLUSIONS: Obtaining routine intrauterine cultures during non-elective cesarean sections might be useful for detecting significant pathogens and tailoring the antibiotic treatment in postpartum endometritis.

Characteristics of hospitalised patients with influenza in 2015-2016 in northern Israel: three circulating strains and continued fear of 2009 A/H1N1.

This study aimed to characterise children and adults diagnosed with influenza who were admitted to three medical centres in northern Israel in the winter of 2015-2016, a unique season due to infection with three types of influenza strains: A/H1N1, A/non-H1N1 and B. Data were collected retrospectively from medical records. Influenza A/H1N1 infected mainly adults (61% vs. 16% in children, P < 0.001) while influenza B was the common type in children (54% vs. 28% in adults, P < 0.001). Adults (36% vs. 5% in children, P < 0.001) and patients infected with A/H1N1 had higher rates of pneumonia (34% vs. 16% and 14% in influenza B and A/non-H1N1, respectively, P = 0.002). Treatment with oseltamivir was prescribed to 90% of patients; adults had higher rates of treatment (96% vs. 84% in children, P = 0.002) as well as patients infected with A/H1N1 (96% vs. 86% in influenza B and A/non-H1N1, respectively, P = 0.04). Oseltamivir was given after a mean of 3.6 days of symptoms. Preferential infection of adults by A/H1N1 was evident in Israel in 2015-2016; pneumonia rates were higher in adults and in A/H1N1-infected patients. Oseltamivir was prescribed to most patients but especially to those infected with A/H1N1, and was given relatively late in the course of the disease.

Analysis of the First 100 Patients From the Syrian Civil War Treated in an Israeli District Hospital.

OBJECTIVE: An analysis of the injuries and treatment of the first 100 patients from the Syrian civil war was conducted to monitor quality of care and outcome. SUMMARY BACKGROUND DATA: As reports of the collapse of health care systems in regions within Syria reach the media, patients find themselves crossing the border into Israel for the treatment of war injuries. Among these patients are combatants, noncombatants, women, and children. Treatment, that is free at the point of care, is a humanitarian imperative for war wounded, and this paper reports the care in an Israeli district hospital of the first 100 patients received. METHODS: With ethics committee approval, data from the Trauma Registry and electronic patient records were collected and analyzed. No identifying data are presented. RESULTS: Most patients (94) were male. Seventeen patients were younger than the age of 18 years; 52 patients were in their twenties. Most injuries were the results of gunshot or blast injury (50 and 29 patients, respectively). Two multiple-trauma patients died, 8 were transferred for specialist care, and 90 patients returned from Ziv Hospital to Syria after discharge. CONCLUSIONS: The experience of the care of patients across a hostile border has been unprecedented. Hospital protocols required adjustment to deliver quality clinical and social care to patients suffering from both the acute and chronic effects of civil war.

Reconsidering the Current Preterm Premature Rupture of Membranes Antibiotic Prophylactic Protocol.

OBJECTIVE: The purpose of our study was to determine whether the current antibiotic regimen for preterm premature rupture of membranes (PPROM) is adequate for covering the current causative agents and sensitivities of chorioamnionitis and early-onset neonatal sepsis. STUDY DESIGN: During a 3-year period, we retrieved the results from placental and amniotic membrane cultures obtained at delivery in cases of maternal fever, chorioamnionitis, and PPROM, and from blood cultures obtained from neonates with early-onset sepsis (EOS) in three participating hospitals. Sensitivity of pathogens to antimicrobial agents was performed using routine microbiologic techniques. RESULTS: There were 1,133 positive placental or amniotic cultures, 740 (65.3%) were from gram-negative Enterobacteriaceae. There were 27 neonates diagnosed with EOS with positive blood cultures. Aerobic Enterobacteriaceae accounted for 14 cases (52%) and group B streptococcus for 7 cases (26%). Of the Escherichia coli and Klebsiella sp., only 38% were sensitive to ampicillin. CONCLUSION: Local pathogens and their antibiotic sensitivity profiles should be explored every few years and an effective antibiotic protocol chosen to cover the main pathogens causing chorioamnionitis and EOS. Consideration should be made for changing ampicillin in women with PPROM to a regimen with better coverage of gram-negative Enterobacteriaceae.

Reconstitution of an active arginine deiminase pathway in Mycoplasma pneumoniae M129.

Some species of the genus Mycoplasma code for the arginine deiminase pathway (ADI), which enables these bacteria to produce ATP from arginine by the successive reaction of three enzymes: arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB), and carbamate kinase (ArcC). It so far appears that independently isolated strains of Mycoplasma pneumoniae encode an almost identical truncated version of the ADI pathway in which the proteins ArcA and ArcB have lost their original enzymatic activities due to the deletion of significant regions of these proteins. To study the consequences of a functional ADI pathway, M. pneumoniae M129 was successfully transformed with the cloned functional arcA, arcB, and arcC genes from Mycoplasma fermentans. Enzymatic tests showed that while the M. pneumoniae ArcAB and ArcABC transformants possess functional arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, they were unable to grow on arginine as the sole energy source. Nevertheless, infection of a lung epithelial cell line, A549, with the M. pneumoniae transformants showed that almost 100% of the infected host cells were nonviable, while most of the lung cells infected with nontransformed M. pneumoniae were viable under the same experimental conditions.

Genomic features and insights into the biology of Mycoplasma fermentans.

We present the complete genomic sequence of Mycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977,524 bp and has a mean G+C content of 26.95 mol%. There are 835 predicted protein-coding sequences and a mean coding density of 87.6 %. Functions have been assigned to 58.8 % of the predicted protein-coding sequences, while 18.4 % of the proteins are conserved hypothetical proteins and 22.8 % are hypothetical proteins. In addition, there are two complete rRNA operons and 36 tRNA coding sequences. The largest gene families are the ABC transporter family (42 members), and the functionally heterogeneous group of lipoproteins (28 members), which encode the characteristic prokaryotic cysteine 'lipobox'. Protein secretion occurs through a pathway consisting of SecA, SecD, SecE, SecG, SecY and YidC. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The genes encoding DnaK-DnaJ-GrpE and Tig, forming the putative complex of chaperones, are intact, providing the only known control over protein folding. Eighteen nucleases and 17 proteases and peptidases were detected as well as three genes for the thioredoxin-thioreductase system. Overall, this study presents insights into the physiology of M. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.

Mycoplasma hyorhinis upregulates calpastatin and inhibits calpain-dependent proteolysis in SH-SY5Y neuroblastoma cells.

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen.

Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.

Reconstituted proteolipid vesicles prepared from Mycoplasma fermentans membranes are able to bind and fuse with Molt-3 cells.

We describe and characterize reconstituted proteolipid vesicles (rPLV) prepared from solubilized Mycoplasma fermentans membranes and studied their binding to and fusion with host Molt-3 cells. The rPLV were prepared following membrane solubilization by Triton X-100 and detergent removal by SM-2 resin beads. The vesicles thus obtained had a rather uniform diameter of about 1 microm and were sealed as monitored by measuring in an assay that measures the quenching by sodium dithionite of a hydrophobic fluorescent probe incorporated into the rPLV membrane. The rPLV adhered to Molt-3 cells and, based on measurements of lipid mixing, fused with the host cells at a similar rate and to about the same extent as intact M. fermentans. Preliminary experiments showed that a chimeric protein, GnRH-PE66, could be encapsulated within these rPLV, opening the way to develop a system for the transfer of high-molecular weight soluble molecules, encapsulated in the rPLV, to target eukaryotic cells.

Recognition of Mycoplasma hyorhinis by CD99-Fc molecule.

The human CD99 protein is expressed on many cell types and is mostly abundant on lymphocytes and on several tumors. Different functions were attributed to the CD99 receptor, including adhesion, apoptosis and activation. However, until now the only ligand suggested to be recognized by CD99 was CD99 itself. In order to identify possible new CD99 ligands we constructed a CD99 protein fused to human IgG1. Surprisingly, a pronounced specific staining of melanoma cell lines that were infected with mycoplasmas was observed whereas clean cells were not recognized. Staining was specific, as other fusion proteins did not recognize the mycoplasma-infected cells. Sequencing of the 23s-16s region revealed that the contaminating agent is Mycoplasma hyorhinis. The CD99 interaction with M. hyorhinis was direct since it was blocked by anti-CD99 monoclonal antibody and by M. hyorhinis. It was also strain-specific as other mycoplasmas were not recognized. Our results show that CD99 interacts with a novel ligand of M. hyorhinis.