Neurologic and cutaneous infection by Clostridium piliforme in a kitten with systemic Tyzzer disease.

Tyzzer disease (TD) is a highly fatal condition of animals caused by Clostridium piliforme and characterized pathologically by enteritis, hepatitis, myocarditis, and occasionally encephalitis. Cutaneous lesions have been reported only rarely in animals with TD, and infection of the nervous system has not been described in cats, to our knowledge. We describe here neurologic and cutaneous infection by C. piliforme in a shelter kitten with systemic manifestations of TD and coinfection with feline panleukopenia virus. Systemic lesions included necrotizing typhlocolitis, hepatitis, myocarditis, and myeloencephalitis. The cutaneous lesions consisted of intraepidermal pustular dermatitis and folliculitis, with necrosis of keratinocytes and ulceration. Clostridial bacilli were identified within the cytoplasm of keratinocytes by fluorescence in situ hybridization, and a PCR assay was positive for C. piliforme. C. piliforme can infect keratinocytes leading to cutaneous lesions in cats with the location suggesting direct contact with contaminated feces as a route of infection.

Clostridium piliforme infection (Tyzzer disease) in horses: retrospective study of 25 cases and literature review.

Tyzzer disease (TD) is caused by Clostridium piliforme, a gram-negative and obligate intracellular bacterium. The disease occurs in multiple species. A triad of lesions, namely colitis, hepatitis, and myocarditis, is described in cases of TD in some species, such as rats and mice. We carried out a retrospective analysis of 25 equine cases with a diagnosis of TD; 24 of 25 cases occurred in foals <45 d old; the remaining foal was 90 d old. There were 12 males and 12 females; no sex information was available for one foal. The affected breeds were Quarter Horse, Thoroughbred, Arabian, Paint, and Hanoverian. Most of the cases (19 of 25) occurred in the spring. There were 9 cases of sudden death; the remaining animals had diarrhea, fever, distended abdomen, depression, weakness, non-responsiveness, and/or recumbency. Gross findings included icterus, hepatomegaly with acinar pattern, serosal hemorrhages, pulmonary edema, and/or fluid content in small and large intestine. Microscopically, all foals had severe, multifocal, necrotizing hepatitis. Necrotizing lymphohistiocytic colitis was observed in 10 of 25 foals, and multifocal necrotizing myocarditis was found in 8 of 25. Gram-negative, Steiner-positive, intracytoplasmic filamentous bacteria were observed in hepatocytes, enterocytes, and myocardiocytes, respectively. PCR detected C. piliforme DNA in the liver (24 of 24), colon (20 of 24), and heart (5 of 25). Our results indicate that necrotic hepatitis is the hallmark of TD in horses; the so-called triad of lesions is not a consistent characteristic of the disease in this species.

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense.

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

Microbiological features, epidemiology, and clinical presentation of Clostridioidesdifficile strains from MLST Clade 2: A narrative review.

Clostridioides difficile is an emerging One Health pathogen and a common etiologic agent of diarrhea, both in healthcare settings and the community. This bacterial species is highly diverse, and its global population has been classified in eight clades by multilocus sequence typing (MLST). The C. difficile MLST Clade 2 includes the NAP1/RT027/ST01 strain, which is highly recognized due to its epidemicity and association with severe disease presentation and mortality. By contrast, the remaining 83 sequence types (STs) that compose this clade have received much less attention. In response to this shortcoming, we reviewed articles published in English between 1999 and 2020 and collected information for 27 Clade 2 STs, with an emphasis on STs 01, 67, 41 and 188/231/365. Our analysis provides evidence of large phenotypic differences that preclude support of the rather widespread notion that ST01 and Clade 2 strains are "hypervirulent". Moreover, it revealed a profound lack of (meta)data for nearly 70% of the Clade 2 STs that have been identified in surveillance efforts. Targeted studies aiming to relate wet-lab and bioinformatics results to patient and clinical parameters should be performed to gain a more in-depth insight into the biology of this intriguing group of C. difficile isolates.

Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense.

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

Clostridium massiliamazoniense sp. nov., New Bacterial Species Isolated from Stool Sample of a Volunteer Brazilian.

The study of the gut microbiota by the "culturomics concept" permitted us to isolate, from human stool sample, an unknown anaerobic bacterium within the genus Clostridium for which we propose the name Clostridium massiliamazoniense sp. nov. It was isolated from the fecal flora of a healthy 49-year-old Brazilian male. Here, we describe the characteristics of this organism and its complete genome sequencing and annotation. Clostridium massiliamazoniense sp. nov., ND2T (= CSURP1360 = DSMZ 27309) is a Gram-positive, obligate anaerobic member of Firmicutes with a 3,732,600 bp-long genome and a G+C content of 27.6%.

Genomic Characterization of Newly Completed Genomes of Botulinum Neurotoxin-Producing Species from Argentina, Australia, and Africa.

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species.

Diet during Pregnancy and Infancy and the Infant Intestinal Microbiome.

OBJECTIVES: To determine the association between diet during pregnancy and infancy, including breastfeeding vs formula feeding, solid food introduction, and the infant intestinal microbiome. STUDY DESIGN: Infants participating in the Vitamin D Antenatal Asthma Reduction Trial were included in this study (n = 323). Maternal and infant diets were assessed by questionnaire. Infant stool samples were collected at age 3-6 months. Stool sequencing was performed using the Roche 454 platform. Analyses were stratified by race/ethnicity. RESULTS: Breastfeeding, compared with formula feeding, was independently associated with infant intestinal microbial diversity. Breastfeeding also had the most consistent associations with individual taxa that have been previously linked to early-life diet and health outcomes (eg, Bifidobacterium). Maternal diet during pregnancy and solid food introduction were less associated with the infant gut microbiome than breastfeeding status. We found evidence of a possible interaction between breastfeeding and child race/ethnicity on microbial composition. CONCLUSIONS: Breastfeeding vs formula feeding is the dietary factor that is most consistently independently associated with the infant intestinal microbiome. The relationship between breastfeeding status and intestinal microbiome composition varies by child race/ethnicity. Future studies will need to investigate factors, including genomic factors, which may influence the response of the microbiome to diet. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00920621.

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

Background: An effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated. Results: Recombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1. Conclusions: Insertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of Clostridium species isolated from Saudi Arabia

Abstract The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha’il region of Saudi Arabia in September 2014 using MALDI–TOF-MS. Clostridium spp. were identified by Bruker MALDI–TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI–TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500 bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI–TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI–TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms.

Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of Clostridium species isolated from Saudi Arabia.

The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha'il region of Saudi Arabia in September 2014 using MALDI-TOF-MS. Clostridium spp. were identified by Bruker MALDI-TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI-TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI-TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI-TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms.

Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.

A vanG-type locus in Clostridium argentinense.

OBJECTIVES: The objective was to study a new vanG-type locus in Clostridium argentinense vanGCar and to determine its impact on glycopeptide susceptibility of the host. METHODS: The whole genome of C. argentinense NCIB 10714 was sequenced using Illumina single-reads sequencing technology. The presence of vanGCar in seven C. argentinense strains was tested by PCR and its expression was tested by quantitative RT-PCR (qRT-PCR). Glycopeptide susceptibility was determined by the Etest procedure. RESULTS: The vanGCar locus contained four genes encoding a carboxypeptidase, a d-alanine:d-serine ligase, a serine transporter and a serine racemase, and was present in the seven C. argentinense studied. An AraC-type transcriptional regulator was found upstream from the genes. C. argentinense NCIB 10714 was susceptible to vancomycin and to teicoplanin. qRT-PCR experiments revealed that vanGCar was not expressed without or with induction by a subinhibitory concentration of vancomycin. CONCLUSIONS: The new vanGCar locus was cryptic in C. argentinense and intrinsic to this species. Emergence of vancomycin resistance in C. argentinense due to decryptification of the vanGCar gene cluster could occur.

New FeFe-hydrogenase genes identified in a metagenomic fosmid library from a municipal wastewater treatment plant as revealed by high-throughput sequencing.

A fosmid metagenomic library was constructed with total community DNA obtained from a municipal wastewater treatment plant (MWWTP), with the aim of identifying new FeFe-hydrogenase genes encoding the enzymes most important for hydrogen metabolism. The dataset generated by pyrosequencing of a fosmid library was mined to identify environmental gene tags (EGTs) assigned to FeFe-hydrogenase. The majority of EGTs representing FeFe-hydrogenase genes were affiliated with the class Clostridia, suggesting that this group is the main hydrogen producer in the MWWTP analyzed. Based on assembled sequences, three FeFe-hydrogenase genes were predicted based on detection of the L2 motif (MPCxxKxxE) in the encoded gene product, confirming true FeFe-hydrogenase sequences. These sequences were used to design specific primers to detect fosmids encoding FeFe-hydrogenase genes predicted from the dataset. Three identified fosmids were completely sequenced. The cloned genomic fragments within these fosmids are closely related to members of the Spirochaetaceae, Bacteroidales and Firmicutes, and their FeFe-hydrogenase sequences are characterized by the structure type M3, which is common to clostridial enzymes. FeFe-hydrogenase sequences found in this study represent hitherto undetected sequences, indicating the high genetic diversity regarding these enzymes in MWWTP. Results suggest that MWWTP have to be considered as reservoirs for new FeFe-hydrogenase genes.

Phenotypic and genotypic characterization of multidrug-resistant Bacteroides, Parabacteroides spp., and Pseudoflavonifractor from a Costa Rican hospital.

Multidrug resistance in Bacteroides spp. and related genera is uncommon and has not been described in Latin America until now. We studied phenotypically and genotypically the multidrug resistance of 10 clinical strains of Bacteroides, two of Parabacteroides distasonis, and one of Pseudoflavonifractor capillosus recovered in a national hospital between 2006 and 2010. To this end, we determined minimum inhibitory concentrations (MICs) of amoxicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, clindamycin, ciprofloxacin, tetracycline, and metronidazole using E-tests, evaluated the isolates for ß-lactamases with nitrocefin hydrolysis tests, performed a polymerase chain reaction (PCR)-based screening of erm, tet, and nim genes, obtained partial gyrA sequences, and studied the effect of tazobactam and efflux pump inhibitors (EPI) on the MIC of cefotaxime, clindamycin, and ciprofloxacin. Three isolates were resistant to four different classes of antibiotics and 10 were resistant to three. ß-lactam resistance was in most cases due to ß-lactamases susceptible of partial inhibition by tazobactam. Ten isolates were cfxA-positive and two isolates had cepA. Twelve isolates were highly resistant to clindamycin and nine were highly resistant to ciprofloxacin. However, these phenotypes were not linked to ermA, ermB, ermF, and ermG or mutations in gyrA. Addition of EPI lowered the MICs of clindamycin and ciprofloxacin of one and four isolates, respectively. Twelve isolates had tetQ and four were positive for tetM. In both cases, genes of the two-component system RteAB accompanied tet genes. Although metronidazole susceptibility was universal, nim genes were not present. To our knowledge, this is the first report of multidrug resistance due to less commonly identified or alternative mechanisms in strains of Bacteroides and related species from a developing country.

Development of a cyclic voltammetry method for the detection of Clostridium novyi in black disease.

Black disease is an acute disease of sheep and cattle. The pathogen is the obligate anaerobe, Clostridium novyi. Due to difficulties of anaerobic culturing in the country or disaster sites, a simple, rapid, and sensitive method is required. In this study, an electrochemical method, the cyclic voltammetry method, basing on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (positive dye, methylene blue), was introduced. DNA extracted from C. novyi specimens was amplified through the LAMP reaction. Then the products combined were with methylene blue, which lead to a reduction in the oxidation peak current (ipA) and the reduction peak current (ipC) of the cyclic voltammetry. The changes of ipA/ipC were real-time measured by special designed electrode, so the DNA was quantitatively detected. The results displayed that this electrochemical detection of C. novyi could be completed in 1-2 h with the lowest bacterial concentration of 10(2) colony forming units/mL, and high accuracy (96.5%), sensitivity (96%), and specificity (97%) compared to polymerase chain reation. The cyclic voltammetry method was a simple and fast method, with high sensitivity and high specificity, and has great potential to be a usable molecular tool for fast diagnosis of Black disease.

Gases and volatile compounds associated with micro-organisms in blown pack spoilage of Brazilian vacuum-packed beef.

UNLABELLED: This study correlated the composition of the spoilage bacterial flora with the main gaseous and volatile organic compounds (VOCs) found in the package headspace of spoiled, chilled, vacuum-packed meat. Fifteen chilled, vacuum-packed beef samples, suffering from blown pack spoilage, were studied using 16S rRNA clone sequencing. More than 50% of the bacteria were identified as lactic acid bacteria (LAB), followed by clostridia and enterobacteria. Fifty-one volatile compounds were detected in the spoiled samples. Although the major spoilage compounds were identified as alcohols and aldehydes, CO2 was identified as the major gas in the spoiled samples by headspace technique. Different species of bacteria contribute to different volatile compounds during meat spoilage. LAB played an important role in blown pack deterioration of the Brazilian beef studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated by this study provided useful information to correlate the microbial contamination of Enterobacteriaceae, lactic acid bacteria and Clostridium with the VOC and gaseous compound production to define, in a faster manner, not only the type of contamination, but also to prevent it.

Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

Establishment of the bacterial fecal community during the first month of life in Brazilian newborns.

OBJECTIVE: The establishment of the intestinal microbiota in newborns is a critical period with possible long-term consequences for human health. In this research, the development of the fecal microbiota of a group of exclusively breastfed neonates living in low socio-economic conditions in the city of São Paulo, Brazil, during the first month of life, was studied. METHODS: Fecal samples were collected from ten neonates on the second, seventh, and 30(th) days after birth. One of the neonates underwent antibiotic therapy. Molecular techniques were used for analysis; DNA was extracted from the samples, and 16S rRNA libraries were sequenced and phylogenetically analyzed after construction. A real-time polymerase chain reaction (PCR) was performed on the samples taken from the 30(th) day to amplify DNA from Bifidobacterium sp. RESULTS: The primary phylogenetic groups identified in the samples were Escherichia and Clostridium. Staphylococcus was identified at a low rate. Bifidobacterium sp. was detected in all of the samples collected on the 30(th) day. In the child who received antibiotics, a reduction in anaerobes and Escherichia, which was associated with an overgrowth of Klebsiella, was observed throughout the experimental period. CONCLUSION: The observed pattern of Escherichia predominance and reduced Staphylococcus colonization is in contrast with the patterns observed in neonates living in developed countries.