Christie, Atkins, Munch-Peterson (CAMP) Negative Listeria monocytogenes Meningitis in Neonates: Report of Two Cases.

Listeria monocytogenes, a gram-positive bacillus and an intracellular pathogen, is an uncommon cause of illness in the general population. During pregnancy, a perinatal infection can lead to serious complications such as abortion, stillbirth, neonatal sepsis, and meningitis. We present two cases of neonatal meningitis caused by Christie, Atkins, Munch-Peterson (CAMP)-negative Listeria monocytogenes. In the first case, a seven-day-old female term neonate delivered vaginally, presented with high-grade fever and refusal to feed. In view of the suspected late-onset sepsis, a septic workup, including cerebrospinal fluid analysis, was conducted. CSF culture reports obtained showed a growth consistent with Listeria monocytogenes, which was CAMP test negative and susceptible to the penicillin group of drugs, cotrimoxazole, erythromycin, and meropenem. The isolate was identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and confirmed by 16S rRNA sequencing. The blood culture was sterile. At 48 hours of admission, the neonate clinically deteriorated with fluctuation in oxygen saturation below 95% at room air. Thus, she was electively intubated and connected to the mechanical ventilator with appropriate settings. The antibiotics were upgraded to meropenem from the empirical antibiotic therapy. The neonate showed clinical improvement within the next 24 hours of initiating antibiotics according to culture susceptibility and was gradually weaned from the mechanical ventilator to continuous positive airway pressure (CPAP). After 24 hours, she was able to maintain normal saturation at room air. In the second case, an 11-day-old low birth weight neonate, small for gestational age, was presented to the NICU with complaints of loose stools, fever, and refusal to feed for the past two days. In view of the suspected sepsis, relevant investigations were carried out while initiating empirical antibiotics IV piperacillin-tazobactam and IV amikacin for the neonate. Meanwhile, there was a dip in oxygen saturation noted on room air for the neonate and he/she was mechanically ventilated. The CSF culture grew Listeria monocytogenes,which was identified using MALDI-TOF MS and confirmed by 16S rRNA sequencing. The isolate tested negative for the CAMP test and was susceptible to ampicillin, penicillin, cotrimoxazole, erythromycin, and meropenem. The blood culture was sterile. The antibiotics were upgraded to meropenem from the empirical antibiotic therapy, the patient's condition improved, and the baby was eventually discharged.

Nonhemolytic Listeria monocytogenes-Prevalence Rate, Reasons Underlying Atypical Phenotype, and Methods for Accurate Hemolysis Assessment.

Listeria monocytogenes is a foodborne pathogen that typically presents ß-hemolytic activity. However, there are literature reports indicating that L. monocytogenes strains are sometimes nonhemolytic or their zones of hemolysis are perceivable only after removal of the colonies from the agar plate. Nonhemolytic L. monocytogenes are most commonly encountered in food products, but some have also been detected in clinical samples. Usually, atypical bacteria of this species belong to serotype 1/2a. Mutations of the prfA gene sequence are the most common reason for changed phenotype, and mutations of the hly gene are the second most common cause. There are also reports that the methodology used for detecting hemolysis may influence the results. Sheep or horse blood, although most commonly used in modern studies, may not allow for the production of clear hemolytic zones on blood agar, whereas other types of blood (guinea pig, rabbit, piglet, and human) are more suitable according to some studies. Furthermore, the standard blood agar plate technique is less sensitive than its modifications such as bilayer or top-layer (overlay) techniques. The microplate technique (employing erythrocyte suspensions) is probably the most informative when assessing listerial hemolysis and is the least susceptible to subjective interpretation.

Is a positive Christie-Atkinson-Munch-Peterson (CAMP) test sensitive enough for the identification of Streptococcus agalactiae?

BACKGROUND: For a long time, the Christie-Atkinson-Munch-Peterson (CAMP) test has been a standard test for the identification of Streptococcus agalactiae, and a positive result for S.agalactiae has been considered sensitive enough. METHODS: To confirm whether a positive CAMP test is a requirement for the identification of S.agalactiae, five suspected CAMP-negative S.agalactiae isolates from two hospitals, confirmed as Gram-positive and catalase-negative streptococci, were verified by the CAMP test in three batches of plates from two manufacturers and identified by the Phoenix system, MALDI-TOF MS, the PCR assay and the 16S rDNA gene sequencing. RESULTS: All five suspected strains were S.agalactiae, four of which were CAMP-negative and one of which was not S.agalactiae by the PCR assay. CONCLUSIONS: A positive CAMP test was lacking sensitivity for the identification of S.agalactiae, and the question of whether the cfb gene is worthy of targeting should be further studied.

Crystal structure of the Streptococcus agalactiae CAMP factor provides insights into its membrane-permeabilizing activity.

Streptococcus agalactiae is an important human opportunistic pathogen that can cause serious health problems, particularly among newborns and older individuals. S. agalactiae contains the CAMP factor, a pore-forming toxin first identified in this bacterium. The CAMP reaction is based on the co-hemolytic activity of the CAMP factor and is commonly used to identify S. agalactiae in the clinic. Closely related proteins are present also in other Gram-positive pathogens. Although the CAMP toxin was discovered more than a half century ago, no structure from this toxin family has been reported, and the mechanism of action of this toxin remains unclear. Here, we report the first structure of this toxin family, revealing a structural fold composed of 5 + 3-helix bundles. Further analysis by protein truncation and site-directed mutagenesis indicated that the N-terminal 5-helix bundle is responsible for membrane permeabilization, whereas the C-terminal 3-helix bundle is likely responsible for host receptor binding. Interestingly, the C-terminal domain inhibited the activity of both full-length toxin and its N-terminal domain. Moreover, we observed that the linker region is highly conserved and has a conserved DLXXXDXAT sequence motif. Structurally, this linker region extensively interacted with both terminal CAMP factor domains, and mutagenesis disclosed that the conserved sequence motif is required for CAMP factor's co-hemolytic activity. In conclusion, our results reveal a unique structure of this bacterial toxin and help clarify the molecular mechanism of its co-hemolytic activity.

Clostridium perfringens - A bacterial pathogen gaining recognition in necrotizing pancreatitis.

We report an interesting case of necrotizing pancreatitis due to Clostridium perfringens in an elderly man who came to the hospital with complaints of severe abdominal pain. The infection further worsened with the dissemination to other internal organs. The patient did not show any improvement despite intensive care and treatment. This emphasizies the fact that early diagnosis and appropriate treatment would reduce the morbidity associated with necrotizing pancreatitis.

Haemolytic differential identification of Arcanobacterium haemolyticum isolated from a patient with diabetic foot ulcers.

INTRODUCTION: Arcanobacterium haemolyticum (formerly known as Corynebacterium haemolyticum) is the causative agent of sore throat and also causes skin and soft tissue infections in diabetes patients. A. haemolyticum is a Gram-positive, catalase-negative, ß-haemolytic bacillus. A. haemolyticum poses a diagnostic challenge in the hospital laboratory because most coryneform bacilli are considered as normal flora or contaminants, and it is therefore difficult to differentiate from ß-haemolytic streptococci by colony characteristics. CASE PRESENTATION: A. haemolyticum was isolated from a diabetic patient with foot ulcers and the isolate was identified by using a VITEK-2 system, CAMP inhibition test, reverse CAMP test and a 23S rRNA gene sequence analysis. The isolated A. haemolyticum inhibited haemolysis of Staphylococcus aureus in the CAMP test and enhanced haemolysis of Streptococcus agalactiae in the reverse CAMP test. The diabetic patient was treated with teicoplanin and imipenem, and the ulcers healed within 2 weeks. CONCLUSION: The present study suggests that a haemolytic differential method using the CAMP inhibition and reverse CAMP tests can be useful for differentiating A. haemolyticum from ß-haemolytic streptococci.

Production and applications of an N-terminally-truncated recombinant beta-haemolysin from Staphylococcus aureus.

The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a ß-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.

Staphylococcus pseudintermedius for CAMP-test.

CAMP test reliably detects Listeria monocytogenes (Lm) and Streptococcus agalactiae (group B streptococcus, GBS); it is traditionally performed streaking the tested isolate perpendicularly to Staphylococcus aureus (Sa), provided that reference Sa strains (that produce ß-hemolysin) are used. In a zone of ß-hemolysin activity, in fact, GBS and Lm form typical arrow-shaped hemolytic areas. While Sa production of the toxin is strain-dependent, however, that of Staphylococcus pseudintermedius (Sp), a pet-owner colonizer and an emerging human pathogen, is constitutive, then observed in all clinical isolates. Therefore, Sp may indeed represent a valid alternative to perform the assay.

Liofilchem(®) O.A. Listeria agar and direct CAMP test provided sooner Listeria monocytogenes identification from neonatal bacteremia.

Listeria monocytogenes infection in pregnant women and newborns is a cause for serious concern, and invasive disease outcome strongly depends on prompt antibiotic therapy. To provide sooner identification from neonatal bacteremia we performed a CAMP test directly on positive blood aliquots and inoculated the Liofilchem(®) O.A. Listeria chromogenic agar as well, thus providing a 24-h turn-around time for response.

Estudio piloto de la prueba de percepción musical camp en implantados y normoyentes / Clinical assesment of the music perception test camp in implant users and normal- hearing subjects: a pilot study

En la búsqueda de una prueba de percepción musical estandarizada y aplicable en español, se evaluó el CAMP test creado en inglés por la Universidad de Washington para implantados y normoyentes. Objetivo: determinar las dificultades de su aplicación cuando el idioma nativo es español. Método: estudio de corte transversal con tres pruebas de percepción musical: umbral de tonos, percepción de melodías y timbre musical. Para correr el programa se tradujeron al español los comandos. Conclusión: la prueba CAMP de percepción musical resulta útil fácil de aplicar en normoyentes y usuarios de implante coclear cuyo idioma nativo sea español, cuando se dispone de cartillas con traducción de los comandos.

The CAMP test, created by the University of Washington for English-speaking implant users and normal-hearing subjects was evaluated in the search to provide a standardized music perception assessment for Spanish-speaking subjects. Objective: to determine administration difficulties when Spanish is the native language. Method: a cross-sectional study developed to examine three aspects of music perception: pitch discrimination threshold, melody recognition responand timbre recognition. Commands were translated into Spanish in order to run the program. Conclusions: The CAMP test for music perception is easily applied in Spanish-speaking normal-hearing individuals and cochlear implant users when instructions translated into Spanish are available.

Modified Spot CAMP Test: A rapid, inexpensive and accurate method for identification of group B streptococci / Prueba de CAMP por spot modificada: Un método rápido, preciso y económico para la identificación de estreptococos grupo B

A rapid modified spot CAMP test using 183 clinical isolates of β haemolytic streptococci was compared with the standard CAMP test described by Christie et al. The scheme of biochemical identification and serological confirmation was taken as reference method. The sensitivity of both tests was 100%, and the specificity of the rapid and standard tests was 96.8% and 88.9% respectively. The modified spot CAMP test is a rapid, inexpensive and accurate method for the identification of group B streptococci, and is more specific than the standard CAMP test.

En este estudio se comparó los resultados de una prueba de CAMP por spot modificada en 20 minutos y la prueba de CAMP original descripta por Christie et al usada para la identificación de Streptococcus agalactiae. Se analizaron 183 aislamientos de estreptococos β hemolíticos, tomando como método de referencia el esquema tradicional de identificación bioquímica y confirmación serológica. La sensibilidad de ambas pruebas fue del 100% y la especificidad de la prueba rápida y la estándar fue de 96,8% y 88,9% respectivamente. La prueba de CAMP por spot modificada es un método rápido, económico y seguro para la identificación de estreptococos del grupo B y posee mayor especificidad que la prueba original.

PLASMID MEDIATED DRUG RESISTANCE IN VIBRIO CHOLERAE 0139 BENGAL.

Sixteen strains of Vibrio cholerae were isolated from cases of diarrhoea. Out of these, 12 (75%) were identified as Vibrio cholerae 0139 synonym Bengal and 4 (25%) as Vibrio cholerae El Tor by standard biochemical and serological tests. Modified CAMP reaction in sheep blood agar showed that 0139 produced moderate hemolysis, El Tor produced wider zone of hemolysis whereas Classical Vibrio cholerae produced no zone of hemolysis (CAMP negative). Break point minimum inhibitory concentration (MIC) by agar dilution method showed that all 0139 strains were resistant to ampicillin 8 mg/L, streptomycin 1 mg/L, chloramphenicol 8 mg/L, sulphamethoxazole 32 mg/L and trimethoprim 0.3-128 mg/L, 58.3% were sensitive to gentamicin 1 mg/L, and all were sensitive to norfloxacin 1 mg/L and cefotaxime 1 mg/L. Resistance to trimethoprim, sulphamethoxazole, ampicillin and gentamicin in 5 strains could be transferred to E coli K-12 by conjugation experiment at a rate of 5×10-6 to 4×10-3. Distinct plasmid bands of 35.8 mega daltons could be seen in agarose gel electrophoresis.