In order to ensure Umbilical Cord Blood Units (UCBU) supply and safety, suitable for transplantation, it is necessary to have strict quality controls. These controls include: CD34+ cell count, typing by Human Leukocyte Antigen (HLA), serological tests, clonogenic capacity, and microbiological monitoring.1–3 Collection, manipulation, cryopreservation, and transplantation of UCBU involve a large number of procedures that are carried out in different areas and can result in microbial contamination of the final UCBU. For the most part, the collection of Cord Blood (CB) is carried out in an operating room with the use of safety procedures such as skin disinfection, closed collection systems, and sterile equipment among others. However, microbial contamination can occur during this process. Besides manipulation, cryopreservation, and thawing provide chances for the introduction of bacteria into UCBU.4 Additionally, several sources of contamination have been described, e.g. vaginal flora, skin, poor aseptic techniques in the areas, use of contaminated material, and others.5,6 It has been reported that the rate of contamination of UCBU ranges from 0 to 48%.7,8 The use of UCBU in sterile conditions is of vital importance because patients requiring this type of transplants are severely immunocompromised. Therefore, these patients have a high risk of contracting infections associated with the transplant. Previous studies have shown that the use of contaminated UCBU is one of the causes of morbidity and mortality in these patients.9 The identification of sources of contamination is crucial, from the sampling of CB until its processing in the laboratory. In previous research, identification of the contaminants was performed by phenotypic characteristics, such as biochemical tests, serotypes, and antimicrobial susceptibility.10,11 More recently, molecular typing methods, such as Enterobacterial Repetitive Intergenic Consensus (ERIC), based on Polymerase Chain Reaction (PCR) “ERIC-PCR”,12,13,14 have replaced previous techniques allowing better epidemiological determination of contamination sources. This molecular method is fast, simple, and highly reproducible. It also has been widely used in a large number of microbial types as well as in the study of clonality and identification of contamination sources. In addition, the new molecular technique for detecting bacteria allows for a quick and easy identification, compared with the classical microbiological methods. In this study we implemented the use of 16s rRNA gene sequencing and ERIC-PCR for typing the microbial strains obtained from criopreservated UCBU in the CB bank (CBB) of the National Center of Blood Transfusion (NCBT) in a period of 11 years (2003–2013) to identify potential sources of microbial contamination and carry out measures for their prevention. Tests of antimicrobial resistance/susceptibility and phenotypic activities that may play an important role in microbial infection were performed for the isolated strains. Implications and consequences of the therapeutic use of UCBU for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

A retrospective analysis over 11 years of cryopreserved UCBU in the CBB of the CNTS was performed. A total of 120 UCBU known to be contaminated by bacteria (aerobic, anaerobic, or both) were identified and extracted from the tank of liquid nitrogen (Bioarchive™ System TG 3626). They were immediately immersed in water bath at 37°C for thawing.

This study reports the type and incidence of bacterial contamination of UCBU for transplant at the CBB of the National Center of Blood Transfusion in Mexico City in a period of 11 years (2003–2013). A total of 5193 CB samples were collected between May 2003 and December 2013. Of those, 1831 (35.25%) were accepted to be cryopreserved and 3, 362 (64.74%) units were excluded for various reasons: weight (suitable volume), white blood cells count (<7×106), total nucleated cell number (<8×108), CD34+ cell number (<2×106), expiration (>48h), doubly reactive serology (HCV, HBV, HIV, Chagas disease, brucellosis, or syphilis), and microbial contamination (aerobic, anaerobic, or both). The incidence of bacterial contamination of UCBU was 2.31%, or 120 units, of which in 13 units bacterial could not be recovered after thawing possibly due to bacterial thermal shocks following exposure to extreme temperatures. The bacterial contamination rate reported in this work are much lower than those reported in previous studies, with microbial contamination rates varying from 0 to 48%.7,8 In this work, a wide variety of bacterial contaminants were isolated and identified from the UCBU. The most frequent bacteria identified were associated with vaginal flora (3.73%), skin (22.44%), and gastrointestinal flora (70.87%). Roseomonas genomospecies 5 (0.93%) was also detected. The advantages of automated culture are: a shortened incubation period (down to 7 days) and rapid availability of positive results due to kinetic monitoring with continuous readout of CO2 production (BacT/ALERT System). However, this automated system focuses exclusively on the cultivation of mesophilic microorganisms, excluding potentially pathogenic bacteria and fungi such as Pseudomonas and Candida since their optimal growth temperature is 28°C. This is important when it comes to nosocomial bacteria that could contaminate CB and thus not be detected by these automated culture systems. Organisms detected in the UCBU were similar to those of other published reports.6,11,18 Gram-negative rods, members of the Enterobacteriaceae family are well known for their ability to cause life-threatening infections in humans. Although they do not normally colonize the skin, they may be present transiently. K. pneumoniae, Escherichia coli (both identified in this work), and other Enterobacteriaceae members have virulence factors such as toxins, adhesins, and capsules that may enable them cope with host defense mechanisms.19–22 Moreover, these organisms may have mobile genetic elements such as plasmids, integrons, and transposons that confer antibiotic resistance. Consequently, eradication of infections with common antimicrobial treatment in immunosuppressed patients become a relevant problem.23,24 This is the first study reporting isolation of Roseomonas genomospecies 5 from UCBU. This microorganism belongs to the Acetobacteriaceae family, is a Gram-negative coccobacilli that develops pink-pigmented colonies. Roseomonas are commonly isolated from middle-age women with one of several underlying conditions, including cancer and diabetes. They are also isolated from blood in association with clinical signs of sepsis (wounds).25 Although this genus of bacteria is not considered a primary human pathogen, it has been isolated as commensal bacteria from immunosuppressed patients and young adults with sexually transmitted diseases.26 Presumably, this organism is associated with cross-contamination in the operating room or infection in the CB donor who could be immunosuppressed and unware of it. Additionally, Gram-positive bacteria such as Staphylococcus spp. and Enterococcus spp. are known to survive after cryopreservation processes due to cell wall characteristics. E. faecalis, is a perianal contaminant commonly associated with the environment during CB collection.27 Poor aseptic techniques could be the reason for the high incidence of this bacterial contaminant in this study. Contamination of UCBU is influenced by multiple factors such as bad collection techniques, exhaustive manipulation of CB, CBB size, and others. In this work, the samples of CB were most likely contaminated during collection, due to inadequate skin aseptic technique, direct contact with fecal bacteria during childbirth, and contact with the vaginal flora. There are several sources of CB contamination because they are collected using different methods. Additionally, childbirth (cesarean or normal) is directly related to the sterility of the UCBU. Skin, equipment of collection, processing, and laboratory equipment such as water baths, incubators, centrifuges, and nitrogen tanks have been shown to be potential sources of contamination.6,8,28 Microbial contamination of CB has been significantly reduced in the past eight years in the CBB, as a result of strategies including disinfection methods, automated detection of microbial contaminants, and others (Table 1). These strategies have been successful because they aimed at reducing microbial contamination originated from regular vaginal biota, enteric and transient skin flora. In order to identify clonal relationship between isolates of the same genus and species, molecular typing assays (ERIC-PCR) were performed. Contamination sources were clearly identified in this work in different sites because even if the identified bacteria belonging to the same genus and species, they presented intergenic variations according to molecular typing assays (Fig. 1). Arrows in the different profiles indicate the intergenic variations in the strains genomes of the same genus and species indicating different contamination sources of UCBU. The use of molecular typing techniques such as RAPD-PCR, ERIC-PCR, DGGE-PCR, and others might identify the source of contamination during CB collection and processing distinguishing transient contaminations from intrinsic contamination and undetected bacteremia in the donor. This molecular tool has been widely used to rule out clonal relationships between bacteria from the same genus and species isolated from a specific area.29,30 Correlation of the antimicrobial resistance pattern and plasmid profiles with clonality in contaminants strains is limited, especially in the hospital environment. Here the use of antibiotics generates great selective pressure for resistance acquisition by means of mobile genetic elements such as plasmids, transposons, integrons, and others. A high proportion of strains resistant to first, second, and third generation antibiotics were identified in this work. Previous work has reported the use of UCBU contaminated with bacteria and recommended administration of antibiotics prior to transplantation. Nevertheless, the risk of infection associated with multi-resistant bacteria to antibiotics persists. Patterns of antibiotic resistance found in isolated strains clearly show molecular mechanisms directly involved in the resistance phenotype. It is well known that prior to UCBU transplantation; the patient who receives antimicrobial treatment and immunosuppressive therapy remains with a potential risk of infection, as the antibiotic therapy may not be effective against multidrug resistant contaminants. Further investigations should aim to elucidate the molecular mechanisms involved in antibiotic resistance in isolates of UCBU isolated in this work. The production of exoenzymes and hemolysins are defense mechanisms of microorganisms during their colonization and pathogenesis. The presence of hemolysins, proteases, amylases, and DNases in strains isolated in UCBU clearly shows the potential risk associated to phenotypically identified virulence factors. Molecular assays have also been used to identify virulence factors.31 Although the incidence of contaminated UCBU found in this work is low, it is recommended to systematically carry out quality control protocols in order to prevent microbial contamination (obtaining the CB in the operating room, CBB processes, and cryopreservation). Recently, a measure to prevent microbial contamination of skin CB associated bacteria has been implemented. This technique involves the deviation and disposal of the first milliliters of CB to eliminate the possibility of entering bacteria into the collection bag. Although there are standardized protocols for the collection, transport, processing, and cryopreservation of CB, it is extremely important to continue with Good Manufacturing Practices that include all of the procedures to obtain UCBU. The need to have a wide genetic diversity of UCBU for transplantation in our country is of great importance and the presence of contaminated UCBU directly affects the genetic diversity of UCBU of CBB.

The authors declare no conflicts of interest.