in gipuzkoa , 27 human listeriosis episodes were reported during january 2013february 2014 . all cases produced sepsis in the patients , except 1 case that produced diarrheal disease in a 34-year - old parturient woman who had undergone a splenectomy .
eleven episodes ( 40.7% ) occurred in pregnant or parturient women , and 8 of the children of these patients were affected : 5 newborns ( 4 of them premature infants ) became ill , 2 pregnancies ended in miscarriage , and 1 infant was stillborn .
( for this study , a pregnancy - associated episode was counted only once , whether the causative strain was isolated from mother , child , or both . ) ten cases ( 37.0% ) occurred among the elderly ( > 70 years of age ) and 6 ( 22.2% ) in adults 4560 years of age . of the 6 patients in the 45- to 60-year age group ,
a total of 29 human l. monocytogenes isolates were available for microbiological characterization : 22 from blood , 3 from placental membranes , 2 from cerebrospinal fluid , and 1 each from stool and dermal exudate .
serotypes were established by agglutination ( listeria - o - antisera , difco ; bd diagnostics , sparks , md , usa ) and by multiplex - pcr ( 4 ) .
the predominant serotypes identified were 1/2b ( n = 17 , 58.6% ) and 4b ( n = 8 , 27.6% ) ; 4 isolates were serotype 1/2a ( 13.8% ) .
pulsed - field gel electrophoresis ( pfge ) was performed by using the restriction endonucleases smai and asci ( 5 ) .
the sts of l. monocytogenes isolates were determined by using the multilocus sequence typing ( mlst ) primers and conditions described by the pasteur institute ( 6 ) .
mlst showed that 16 of 17 serotype 1/2b isolates we tested were st87 ; the remaining serotype 1/2b isolate was st3 .
only 2 human l. monocytogenes st87 serotype 1/2b isolates , both from japan , were listed in the pasteur institute mlst database ( 6 ) .
further , pfge showed 2 large clusters within serotype 1/2b , and epidemiologic research detected 2 main outbreaks .
all listeriosis episodes during the study period that were caused by isolate types other than serotype 1/2b and st87 were sporadic .
in the first case cluster ( first outbreak ) , 5 episodes were detected during august 
all isolates from 1 woman at 28 weeks gestation , 1 parturient woman and her newborn child , and 2 newborn twins ( no samples from the mother were available ) showed the same pfge pattern with the restriction enzyme asci ( arbitrarily named as pattern a ) and were st87 ( figure ) .
another 2 l. monocytogenes isolates from cases apparently not related to this first outbreak , isolated in january 2012 and in april 2013 from a 53-year - old man with meningitis and an 84-year - old woman , respectively , showed the same pfge pattern .
pulsed - field gel electrophoresis patterns of 6 listeria monocytogenes serotype 1/2b isolates collected from listeriosis patients in gipuzkoa , northern spain , during january 2013february 2014 .
lanes 1 and 2 , isolates from first outbreak ( pattern a ) ; lanes 3 , 4 , and 5 , isolates from second outbreak ( pattern b ) , including ( lane 5 ) 1 isolate from a food product of foie gras ; lane 6 , l. monocytogenes sequence type 3 serotype 1/2b isolate , unrelated to outbreaks .
for this first outbreak , epidemiologic interviews were conducted starting with the cases observed during september 2013 .
patients were asked about their alimentary habits , focusing on consumption of raw or semicooked animal or animal derivate products .
although no specific food was identified as the possible source of the outbreak , all but 1 of the patients remembered eating cooked ham bought in the butcher s department of a certain supermarket chain located in different villages of the province .
were investigated by pcr and culture after selective enrichment ( 24 listeria enrichment broth ; oxoid , basingstoke , uk ) ) . in the microbiological investigation ( 7 )
, 1 of 6 brands of cooked ham studied grew l. monocytogenes at a low level , but the sample strain was not the serotype 1/2b identified in the outbreak .
no listeria spp . were identified in the remaining 5 brands of cooked ham studied . in the second outbreak ,
10 episodes were reported during early november 2013 through late february 2014 ; patients were 5 elderly persons , 4 previously healthy parturient women , and 1 parturient women who had undergone a splenectomy .
no isolates could be obtained from the mother of an infected newborn and from a miscarriage .
the 10 isolates available for study ( 1 per episode ) were of the same serotype and sequence type , 1/2b and st87 , as isolates from the first outbreak , but these isolates differed in pfge asci pattern ( this pattern arbitrarily named pattern b ) .
isolates from these 2 outbreaks had the same pfge pattern when the smai enzyme was used ( figure ) .
after the epidemiologic survey for this outbreak , we conducted microbiological analysis of several foods .
a foie gras product kept by a patient in his refrigerator yielded a positive culture of l. monocytogenes that had the same phenotype and molecular profile as isolates from the second outbreak .
the presence of the outbreak strain was confirmed in high amounts ( 5.2  10 cfu / g ) by several other cultures from 3 unopened samples from the same brand of foie gras .
after this food source was identified as the source of this second outbreak , all patients from the first outbreak were specifically interviewed again about the consumption of this product , but none remembered having eaten it .
l. monocytogenes infection is serious and has high fatality rates . among the 35 persons infected in this region of spain since 2013 , a total of 6 deaths have occurred : 3 adults , 2 fetuses ( miscarriages ) , and 1 child ( stillbirth ) . without rapid case detection and early treatment ,
l. monocytogenes infections mainly affect elderly persons , pregnant women , newborns , and immunocompromised adults ( 810 ) .
humans are usually infected after eating contaminated food , although the source of the infection is infrequently detected in sporadic cases . for outbreaks , after the epidemiologic survey , a food is usually implicated as the source of infection , but it is not always possible to obtain microbiological confirmation ( 11 ) . in this study , the strain ( same phenotype and genotype ) that caused the second outbreak was obtained from a recently consumed food in the home and in several unopened samples from the same batch of food . however , this source could not be identified as related to the first outbreak .
pregnant women were frequently infected during these outbreaks ; epidemic clones infected 8 pregnant women and their offspring . however , during 2012 and 2013 , serotype 4b isolates of different genotypes also infected 5 pregnant women , resulting in 1 miscarriage , 3 premature newborns , and 1 stillborn infant and 1 premature newborn in a twin pregnancy . in summary , we defined 2 epidemiologically unrelated outbreaks of listeriosis caused by a rare type of l. monocytogenes that occurred at the same time in a small region of spain .
management of this frequently fatal disease requires careful investigation of the source of infection to stop its spread and prompt treatment of infected persons to prevent severe illness and death .
 in the province of gipuzkoa , spain ( 700,000 inhabitants ) , 712 episodes of human listeriosis were recorded annually during 20092012 . however , during january 2013february 2014 , 27 episodes were detected , including 11 pregnancy - associated cases . 
 fifteen cases in 2 epidemiologically unrelated outbreaks were caused by a rare type of listeria monocytogenes , sequence type 87 serotype 1/2b . 