Deyse Christina Vallim

A cluster of Listeria monocytogenes infections in hospitalized adults

BACKGROUND Listeriosis occurs mainly in persons at extremes of age and with immunocompromising conditions. It is believed that most cases of listeriosis are acquired in the community. A cluster of listeriosis in hospitalized patients prompted the present investigation. METHODS We conducted a case series study of listeriosis from August 21, 2006, to June 1, 2007, in a hospital in the city of Rio de Janeiro, Brazil. RESULTS Six patients with Listeria monocytogenes infection were identified: 5 during hospitalization and 1 at a day clinic. By the time the infection was diagnosed, 5 patients had been in the hospital for a mean of 9 days. All patients were elderly (median age, 80 years) and had immunocompromising conditions. Five (83%) patients died. Four patients developed bloodstream infections, 3 caused by serotype 1/2b. Two patients had peritonitis: one caused by serotype 3b and another by serotype 1/2b. Four L monocytogenes isolates belonged to a single pulse-field gel electrophoresis genotype, suggesting a common source. An epidemiologic investigation pointed to the hospital kitchen as the possible contamination. CONCLUSION Data suggest a health care-associated outbreak of listeriosis and highlight the importance of developing guidelines for prevention and treatment of health care-associated foodborne diseases, especially in hospitals with immunocompromised adult patients.

Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil

INTRODUCTION Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC). RESULTS Among the strains tested, serovar L4b (60.3%) was the most prevalent, followed by serovar 1/2a (20.6%), 1/2b (13.2%) and the more uncommon serovars 1/2c, 3b and 4ab (5.9%). All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5%) showed resistance to rifampin, and two (3%) were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2 μg/ml reinforce the need for microbiological surveillance. CONCLUSIONS The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

Occurrence and antimicrobial resistance patterns of Listeria monocytogenes isolated from vegetables

Although the consumption of fresh and minimally processed vegetables is considered healthy, outbreaks related to the contamination of these products are frequently reported. Among the food-borne pathogens that contaminate vegetables is Listeria monocytogenes, a ubiquitous organism that exhibits the ability to survive and multiply at refrigerated temperatures. This study aimed to evaluate the occurrence of L. monocytogenes in vegetables as well as the antimicrobial resistance of isolates. The results showed that 3.03% of samples were contaminated with L. monocytogenes, comprising 2.22% of raw vegetables and 5.56% of ready-to-eat vegetables. Multiplex PCR confirmed the virulence potential of the isolates. Antimicrobial resistance profiling showed that 50% of the isolates were susceptible to the antibiotics used. The resistance of one isolate to penicillin G, a commonly employed therapeutic agent, and the presence of serotype 4b, a serotype commonly associated with food-borne outbreaks, could be potential health hazards for consumers.

Twenty Years of Listeria in Brazil: Occurrence of Listeria Species and Listeria monocytogenes Serovars in Food Samples in Brazil between 1990 and 2012

Listeria spp. isolated from different food products and collected from 12 Brazilian states were sent to the Laboratory of Bacterial Zoonoses (Oswaldo Cruz Institute, Brazil) for identification. The aims of this study were to characterize these isolates, from 1990 to 2012, by using biochemical, morphological, and serotyping tests, and to analyze the distribution of L. monocytogenes serotypes on different food products and geographical locations. Serotyping was performed using polyclonal somatic and flagellar antisera. Of 5953 isolates, 5770 were identified as Listeria spp., from which 3429 (59.4%) were L. innocua, 2248 (38.9%) were L. monocytogenes, and 93 (1.6%) were other Listeria spp. L. innocua was predominantly isolated from 1990 to 2000, while L. monocytogenes was from 2001 to 2012. Regarding the serotype distribution in the foods, serotypes 1/2a and 4b were most common in processed meat and ready-to-eat products, respectively; serotypes 1/2a, 1/2b, and 4b were the most common in nonprocessed meat. The results above confirm the presence of the main serotypes of L. monocytogenes in different parts of the food chain from three regions of the country and emphasize the importance of improving the control measures, as tolerance zero policy and microbiological surveillance in Brazil.

Serotypes and Pulsotypes Diversity of Listeria monocytogenes in a Beef-Processing Environment

Utensils and equipment from meat-processing facilities are considered relevant cross-contamination points of Listeria monocytogenes to foods, demanding tracking studies to identify their specific origins, and predict proper control. The present study aimed to detect L. monocytogenes in a beef-processing facility, investigating the diversity of serotypes and pulsotypes in order to identify the possible contamination routes. Surface samples from knives (n=26), tables (n=78), and employees hands (n=74) were collected before and during the procedures from a beef-processing facility, in addition to surface samples of end cuts: round (n=32), loin (n=30), and chuck (n=32). All samples were subjected to L. monocytogenes screening according ISO 11.290-1, and the obtained isolates were subjected to serotyping and pulsed-field gel electrophoresis. Listeria spp. were identified in all processing steps, in 61 samples, and L. monocytogenes was detected in 17 samples, not being found only in knives. Eighty-five isolates were identified as L. monocytogenes, from serotypes 1/2c (n=65), 4b (n=13), and 1/2b (n=7), being grouped in 19 pulsotypes. Considering these results, cross-contamination among hands, tables, and beef cuts could be identified. The obtained data indicated the relevance of cross-contamination in the beef-processing facility, and the occurrence of serotypes 1/2b and 4b in beef cuts distributed for retail sale is a public health concern.

Phenotypic and genotypic analysis of bio-serotypes of Yersinia enterocolitica from various sources in Brazil

INTRODUCTION Yersinia enterocolitica is a well-known foodborne pathogen widely distributed in nature with high public health relevance, especially in Europe. METHODOLOGY This study aimed to analyze the pathogenic potential of Y. enterocolitica isolated strains from human, animal, food, and environmental sources and from different regions of Brazil by detecting virulence genes inv, ail, ystA, and virF through polymerase chain reaction (PCR), phenotypic tests, and antimicrobial susceptibility analysis. Pulsed-field gel electrophoresis (PFGE) was used for the assessment of phylogenetic diversity. RESULTS All virulence genes were detected in 11/60 (18%) strains of serotype O:3, biotype 4 isolated from human and animal sources. Ten human strains (4/O:3) presented three chromosomal virulence genes, and nine strains of biotype 1A presented the inv gene. Six (10%) strains were resistant to sulfamethoxazole-trimethoprim, seven (12%) to tetracycline, and one (2%) to amikacin, all of which are used to treat yersiniosis. AMP-CEF-SXT was the predominant resistance profile. PFGE analysis revealed 36 unique pulsotypes, grouped into nine clusters (A to I) with similarity ≥ 85%, generating a diversity discriminatory index of 0.957. Cluster A comprised all bio-serotype 4/O:3 strains isolated from animal and humans sources. CONCLUSIONS This study shows the existence of strains with the same genotypic profiles, bearing all virulence genes, from human and animal sources, circulating among several Brazilian states. This supports the hypothesis that swine is likely to serve as a main element in Y. enterocolitica transmission to humans in Brazil, and it could become a potential threat to public health as in Europe.

Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR

We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n = 12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819-3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537-5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay.

Characterization of epidemic clones of Listeria monocytogenes serotype 4b isolated from humans and meat products in Brazil

INTRODUCTION Listeria monocytogenes is an important foodborne pathogen and the 4b serotype is responsible for many cases of human listeriosis reported in Brazil. Several listeriosis outbreaks worldwide have involved a small number of well-defined clonal groups, designated as epidemic clones (ECs). METHODOLOGY We studied 71 strains of serotype 4b, including 25 isolates from human cases of listeriosis and 46 from meat-based foods, collected in Brazil between 1977 and 2010. The presence of ECs (I and II) markers and virulence genes (inlA, inlB, ilnC, inlJ and actA) were evaluated by PCR assay. The genetic relationship of ECs-positive strains was assessed by pulsed field gel electrophoresis. RESULTS ECI and ECII markers were found both in human and food strains, with 19.7% positive for the ECI marker and 40.8% for ECII. Most strains (97.2%) were positive for the virulence genes that were studied. Nevertheless, the actA gene amplicons showed two distinct sizes, with all ECI positive strains exhibiting a 105bp deletion. Pulsed field gel electrophoresis (PFGE) analysis allowed the recognition of highly related strains, particularly from two outbreaks of neonatal listeriosis in São Paulo State occurred in 1992 and 1997, both ECII-positive; and two ECI strains from a human case (1982) and from bovine meat (2009). CONCLUSIONS The presence of ECs among clinical samples and beef isolates of serotype 4b from some regions of Brazil highlights the need for rigorous control of production procedures. Furthermore, the association of ECII with two nosocomial outbreaks suggests its ability to spread in these settings.

Virulence genes and genetic relationship of L. monocytogenes isolated from human and food sources in Brazil

The herein presented assay provided a bacteriological and molecular characterization of 100 samples of L. monocytogenes isolated from human (43) and food (57) sources, from several regions of Brazil, and collected between 1975 and 2013. Antigenic characterization defined 49% of serotype 4b samples, followed by 28% of serotype 1/2b, 14% of serotype 1/2c, 8% of serotype 1/2a, and 1% of serotype 3b. Both type of samples from human and food origin express the same serotype distribution. Multiplex PCR analysis showed 13 strains of type 4b with the amplification profile 4b-VI (Variant I). Virulence genes hly, inlA, inlB, inlC, inlJ, actA, plcA, and prfA were detected in all samples, highlighting a deletion of 105pb on the actA gene in 23% of serotype 4b samples. Macrorestriction profile with ApaI at PFGE showed 55 pulsotypes, with the occurrence of the same pulsotype in hospitalized patients in São Paulo in 1992 and 1997, and two other highly related pulsotypes in patients hospitalized in Rio de Janeiro in 2008. Recognized pulsotypes in listeriosis cases have also been detected in food. Thus, the prevalence of a serotype and the persistence of certain pulsotypes herald future problems.

Escherichia coli O26 and O113:H21 on Carcasses and Beef from a Slaughterhouse Located in Mato Grosso, Brazil

Shiga toxin-producing Escherichia coli (STEC) is a group of emerging pathogens that can cause human diseases, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Monitoring slaughtering stages and checking contamination points are crucial for the production of safe food. In this context, the aim of this study was to verify contamination by STEC strains, to determine the contamination points and evaluate the resistance profile to 12 antimicrobials used in both veterinary and human medicine. A total of 80 samples were obtained from eight collection points (pen floor, rectum, hide, carcass swabs and esophagus, diaphragm, masseter, and retail beef tissue samples). The isolates were collected by dilution plating on MacConkey agar with sorbitol, cefixime, and tellurite and analyzed by multiplex polymerase chain reaction for virulence genes. Serotyping of non-O157 was performed, and testing for 12 antibiotics by disk diffusion was carried out. A total of 18 STEC strains were isolated, presenting different virulence profiles. Contamination by STEC was observed in the rectum (5/18), carcass surface (5/18), hide (3/18), diaphragm (2/18), retail beef (2/18), and masseter muscle (1/18). Pen floor swabs and esophagus tissues showed no STEC contamination. Moreover, three strains were identified as O26 and three as O113:H21 strains, which have been linked to HUS and HC outbreak cases in Brazil. All STEC isolates were susceptible to all evaluated antimicrobials, except streptomycin. The presence of STEC strains is a direct risk to the consumer, especially when isolated from retail beef, and contamination can occur during different slaughter stages. However, antimicrobial resistance profiles did not identify multidrug-resistant strains, limiting potential antimicrobial resistance transmission to other pathogens.