PFGE Pulsotype Research Articles

High clonal diversity of Staphylococcus aureus isolates from children's playgrounds in Hungary.

Staphylococcus aureus is one of the most important human pathogenic bacteria and environmental surfaces play an important role in the spread of the bacterium. Presence of S. aureus on children's playgrounds and on toys was described in international studies, however, little is known about the prevalence and characteristics of S. aureus at playgrounds in Europe. In this study, 355 samples were collected from playgrounds from 16 cities in Hungary. Antibiotic susceptibility of the isolates was tested for nine antibiotics. Presence of virulence factors was detected by PCR. Clonal diversity of the isolates was tested by PFGE and MLST. The overall prevalence of S. aureus was 2.81% (10/355) and no MRSA isolates were found. Presence of spa (10), fnbA (10), fnbB (5), icaA (8), cna (7), sea (2), hla (10), hlb (2) and hlg (6) virulence genes were detected. The isolates had diverse PFGE pulsotypes. With MLST, we have detected isolates belonging to ST8 (CC8), ST22 (CC22), ST944 and ST182 (CC182), ST398 (CC398), ST6609 (CC45), ST3029 and ST2816. We have identified a new sequence type, ST6609 of CC45. S. aureus isolates are present on Hungarian playgrounds, especially on plastic surfaces. The isolates were clonally diverse and showed resistance to commonly used antibiotics. These data reinforce the importance of the outdoor environment in the spread for S. aureus in the community.

Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain.

In 2014-2015, the main CTX-M-15- and OXA-48-producing clone in our region was ST15. Recently, K. pneumoniae ST15 isolates co-producing VIM-1 and CTX-M-15 were detected in several hospitals. The aim was to study the emergence and acquisition of this carbapenemase. Between 2017 and 2019, four hospitals submitted twenty-nine VIM-1- and CTX-M-15-producing K. pneumoniae ST15 isolates to our laboratory. Seven representatives of each XbaI PFGE pulsotype were sequenced using short- and long-read technologies. RAST, CGE databases, and Pathogenwatch were used for resistance determinants and capsule-type analysis. Plasmid comparison was performed with Easyfig2.1. Phylogenetic analysis included other contemporary ST15 isolates from Spain. The 29 isolates were clustered into seven different pulsotypes. The selected genomes, from three hospitals in two different provinces, were clustered together (fewer than 35 alleles) and differed by more than 100 alleles from other ST15 isolates obtained in the region. These seven isolates harbored one IncR plasmid (200-220 kb) with a common backbone and four regions flanked by IS26: one contained blaVIM-1, another contained blaCTX-M-15, the third contained blaOXA-1, and the fourth harbored heavy-metal-tolerance genes. The two initial plasmids, from two different centers, were identical, and rearrangement of four regions was observed in the five subsequent plasmids. Our findings showed the first intercenter dissemination of IncR plasmids carrying blaVIM-1, blaCTX-M-15, and metal-tolerance genes mediated by a new lineage of K. pneumoniae ST15. Two different capture events of the blaVIM-1 gene or different IS26-mediated plasmid rearrangements from a common ancestor may explain plasmid variations.

Genomic Evidence for Direct Transmission of mecC-MRSA between a Horse and Its Veterinarian

Methicillin-resistant Staphylococcus aureus bearing the mecC gene (mecC-MRSA) has been reported from animals and humans in recent years. This study describes the first mecC-MRSA isolates of human and equine origin in Hungary (two isolates from horses and one from a veterinarian, who treated one of the infected horses, but was asymptomatic). MRSA isolates were identified by cultivation and PCR detection of the species-specific spa gene and mecA/mecC methicillin resistance genes. The isolates were characterized by antibiotic susceptibility testing, MLST, spa, SCCmec typing, PFGE and whole genome sequencing (WGS). All three isolates belonged to the ST130-t843-SCCmec XI genotype, and carried the mecC and blaZ genes. Apart from beta-lactam drugs, they were sensitive to all tested antibiotics. The isolates of the infected horse and its veterinarian had the same PFGE pulsotype and showed only slight differences with WGS. Hence, this is the first description of direct transmission of a mecC-carrying MRSA between a horse and its veterinarian. The emergence of mecC in the country highlights the importance of the appropriate diagnostics in MRSA identification.

Karbapenemaz Üreten Klebsiella pneumoniae İzolatlarının Hastanemizde Yayılımı: Moleküler Tiplendirme ve Klonal İlişkinin Araştırılması

Objective: Carbapenem resistance has been reported with increasing frequency among members of Enterobacterales, especially in the last 10 years. Screening and detection of carbapenemase-producing isolates is important in terms of both directing the treatment and preventing its spread. In our study, it was aimed to determine the carbapenemase types and molecular epidemiological relationships of carbapenem resistant Klebsiella pneumoniae isolates, which were isolated sequentially from the samples sent to microbiology laboratory of our hospital. Method: A total of 32 carbapenem-resistant K. pneumoniae isolates of the samples sent to microbiology laboratory between July and September 2014, were included in the study. In addition to classical methods, identification of isolates at species level was made with BD Phoenix ID/AST automated system. Carbapenemase types (blaOXA-48, blaNDM, blaIMP, blaKPC, blaVIM and blaGES) of the isolates were investigated by PCR. The clonal relationship between the isolates was assessed with PFGE. Results: It was noted that 18 isolates were obtained from intensive care units, 9 from inpatient and 5 from outpatient departments. The blaOXA48 gene was found in all isolates while the other carbapenemase genes were not found. It was determined that strains were isolated from 32 patients in our hospital had 12 different PFGE pulsotypes, named as A-L. Among these, the most common ones were B (n=18) and closely related B1 pattern (n=2). The remaining isolates were represented by 11 different types. It was observed that the first isolate with B pulsotype was responsible for the spread of the outbreak from General Intensive Care Unit. Conclusion: It has been thought that the spread of carbapenem- resistant K. pneumoniae isolates in the hospital was probably occurred through the transfer of isolates from patients with gastrointestinal colonization to other patients through hospital staff. Therefore, the spread of the isolates in hospitals can be limited by detecting colonization with active surveillance programs and by applying contact isolation and effective infection control measures.

Early Detection and Control of Methicillin resistant Staphylococcus aureus Outbreak in an Intensive Care Unit.

Although Methicillin resistant Staphylococcus aureus (MRSA) is one of the major pathogens of healthcare associated infections, we had only sporadic cases in our intensive care unit (ICU) for years. To investigate the sudden increase in the number of MRSA cases in ICU. Descriptive study. From the 5th December 2016 to 26th January 2017, we detected 11 new MRSA cases in ICU. Screening of 73 ICU healthcare workers (HCWs) and screening of 13 patients was performed for outbreak investigation. Nine clinical isolates available in stocks and eight screening MRSA isolates were included in molecular studies. PFGE, spa-mecA-mecC-PVL in-house multiplex PCR assay and spa typing, SCCmec typing were performed for all isolates. Sequence type of the representative strain was determined by Multi-Locus Sequence typing (MLST). All strains were mecA positive, PVL negative, and have the same antimicrobial susceptibility pattern except for two strains. All clinical, two patient screening and three nasal isolates of HCWs showed the same pulsotype, named clone A. The spa type of outbreak isolates is t030 and the SCCmec type is SCCmecIII; the MLST type of representative strain is ST239 (PFGE pulsotype A, ST239-SCCmecIII-t030). Unrelated three isolates had PFGE pulsotype B-SCCmecI-t030, PFGE pulsotype C-SCCmecIII-t459, PFGE pulsotype D-SCCmecIII. Molecular typing techniques are the cornerstones for the investigation of outbreaks. Infection control measures, such as enhancing cleaning procedures, promoting hand hygiene, should be enforced in the ICU unit. All patients, including those who have already been discharged to other departments, must be put on contact isolation. HCWs carrying the MRSA strains could be offered decolonization.

Prevalence of methicillin-resistant Staphylococcus aureus colonization among healthcare workers at a tertiary care hospital in northeastern Brazil

SummaryBackgroundStaphylococcus aureus is a human pathogen of clinical importance related to a variety of infections.AimThe objective of this study was to analyze the molecular and epidemiological characteristics of S. aureus obtained from healthcare professionals (HCP) of a hospital in southwestern Bahia, Brazil.MethodsSamples were collected from hands, nasal cavity, and laboratory coats of 80 HCP. The bacterial isolates recovered from 240 samples were identified as S. aureus, and then analyzed for their antimicrobial resistance profile, genotypic characterization, and pathogenicity.Findings178 isolates were identified as S. aureus, being mostly isolated from the nasal cavity. Thirty isolates (16.8%) were characterized as MRSA. The virulence gene frequency varied according to isolate source. All virulence genes were identified in at least one hand isolate. Isolates from laboratory coats did not show seb and pvl. Isolates from the nasal cavity did not exhibit pvl. The SCCmec type I was identified in 56.7% of MRSA isolates. Among MRSA isolates, 14 PFGE pulsotypes were characterized, with profile A being predominant (nine isolates). Clonal complexes CC5, CC45, and CC398 were found. MRSA isolates induced cytokine gene expression in macrophages, with IL-10 and IL-17 being expressed more often.ConclusionWe found a high colonization rate for S. aureus among HCP. Moreover, we observed that MRSA strains presented different virulence factors and could induce cytokine gene expression, indicating an urgent need to control colonization rates of HCP by MRSA isolates in order to protect hospital patients and the general public.

A report on the first outbreak of emm89 group A streptococcus invasive infections in a burns unit in Tunisia.

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone

BackgroundPseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.ResultsIn a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241 bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2″)-Ia, two Tn402-like with ant(3″)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.ConclusionThis is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

Clonal relationship and the association of the ST218 strain harboring blaOXA-72 gene to mortality in carbapenem-resistant Acinetobacter baumannii bacteremia.

In 2017, the World Health Organization categorized carbapenem-resistant Acinetobacter baumannii (CRAB) as a priority 1, critical antibiotic-resistant bacteria. This study analyzed the clinical outcomes and investigated the molecular epidemiology of CRAB bacteremia in a medical center in Northern Taiwan. We collected 62 blood isolates from patients with CRAB bacteremia from January 2014 to December 2015at MacKay Memorial Hospital and determined the clonal relationship using the PCR-based technique for molecular epidemiology. Medical charts were reviewed for clinical outcomes. Fifty-six isolates harbored the blaOXA-51-like and blaOXA-23-like carbapenemase genes, 4 isolates harbor the blaOXA-51-like and blaOXA-24-like carbapenemase genes and 2 isolates harbored only the blaOXA-51-like gene. After sequencing, all four isolates of blaOXA-24-like carbapenemase gene were confirmed to be isolates of blaOXA-72 carbapenemase genes. In multivariate analysis in the 60 patients, the independent mortality risk factors of CRAB bacteremia included ≥65 years (elderly) (Odds ratio, 4.04, 95% CI, 1.10-14.83, p=0.035), chronic kidney disease (4.36, 1.14-16.72, p=0.032). Isolates harboring the blaOXA-72 gene had the same sequence type (ST218) and PFGE pulsotype raising the possibility of intra-hospital transmission, and all infected patients died. This study showed the clonal relationship of isolates harboring the carbapenemase gene in CRAB bacteremia. Patients with the ST218 strain harboring blaOXA-72 gene had high mortality. This warrants further research to determine the mechanism of virulence and risk factors in order to reduce mortality.

Effectiveness of a divalent Streptococcus dysgalactiae inactivated vaccine in cobia (Rachycentron canadum L.)

Streptococcus dysgalactiae is a major concern in cobia (Rachycentron canadum) cultured in Taiwan; however, there have been no reports on vaccines against this pathogen. This study considered the immune response and protective efficacies of a S. dysgalactiae-derived inactivated vaccine in this fish species. Streptococcus dysgalactiae isolate SD49, belonging to the predominant PFGE pulsotype S1A1, in which 86% of isolates have been classified and SD12 being the highest virulent strain were two vaccine candidates chosen for this study. The bacterin vaccine using a mixture of these two isolates was safe and immunized fish induced greater lysozyme, phagocytosis activities, and antibody responses. Notably, the vaccine protected cobia from the highest virulent S. dysgalactiae strain with a 100% relative percentage survival when challenged 6 weeks post primary vaccination. Together, the divalent vaccine could stimulate immune responses and effectively protect cobia against S. dysgalactiae infection.

Variation in Daily Shedding Pattern of Staphylococcus aureus in Naturally Occurring Intramammary Infections

The cost of mastitis has been calculated at 6% of the value of production; at 2007 prices that is upwards of 2.1 billion dollars. It is estimated that 70-80% of this loss is due to subclinical intramammary infections (IMI) caused by organisms such as Staphylococcus aureus (SA). The control of SA is contingent on accurate diagnosis of IMI, yet currently there exists no standard for the diagnosis of SA IMI. As a result, comparisons between published works are difficult. In addition, the shedding of SA from infected quarters has been described as "intermittent", resulting in recommendations for the diagnosis of SAIMI that are cumbersome and cost-prohibitive in veterinary practice and field research. The goal of this study was to describe shedding patterns of naturally occurring SA IMI over an extended period of time and to provide a reasonable foundation upon which to determine appropriate diagnostic criteria for SAIMI. The effect of PFGE pulsotype on shedding was also examined.

A new mosaic integrative and conjugative element fromStreptococcus agalactiaecarrying resistance genes for chloramphenicol (catQ) and macrolides [mef(I) anderm(TR)

To investigate the genetic basis of catQ-mediated chloramphenicol resistance in Streptococcus agalactiae. Two clinical strains of catQ-positive chloramphenicol-resistant S. agalactiae (Sag236 and Sag403) were recently isolated, typed (MLST, PFGE pulsotypes, capsular types) and their antibiotic resistances investigated by phenotypic and genotypic approaches. Several molecular methods (PCR mapping, restriction assays, Southern blotting, sequencing and sequence analysis, conjugal transfer assays) were used to determine the genetic context of catQ and characterize a genetic element detected in the isolates. Sag236 and Sag403 shared the same ST (ST19), but exhibited a different capsular type (III and V, respectively) and pulsotype. Both harboured the macrolide resistance genes mef(I) and erm(TR) and the tetracycline resistance gene tet(M). Accordingly, they were resistant to chloramphenicol, erythromycin and tetracycline. catQ and mef(I) were associated in an IQ module that was indistinguishable in Sag236 and Sag403. In mating assays, chloramphenicol and erythromycin resistance proved transferable, at low frequency, only from Sag236. Transconjugants carried not only catQ and mef(I), but also erm(TR), suggesting a linkage of the three resistance genes in a mobile element, which, though seemingly non-mobile, was also detected in Sag403. The new element (designated ICESag236, ∼110 kb) results from recombination of two integrative and conjugative elements (ICEs) originally described in different streptococcal species: S. agalactiae ICESagTR7, carrying erm(TR); and Streptococcus pneumoniae ICESpn529IQ, carrying the prototype IQ module. These findings strengthen the notion that widespread streptococcal ICEs may form mosaics that enhance their diversity and spread, broaden their host range and carry new cargo genes.

Foodborne transmission of Listeria monocytogenes via ready-to-eat salad: A nationwide outbreak in Switzerland, 2013–2014

From 26 October 2013 to 23 April 2014, 32 cases of listeriosis infected with an Listeria monocytogenes strain serovar 4b, sequence type 4 and belonging to a single distinct PFGE pulsotype were registered in patients from several cantons of Switzerland. L. monocytogenes was detected in blood (75%), CSF (16%), ascites (6%) and in joint fluid (3%) samples. By the end of March 2014, a food producing company reported an L. monocytogenes contamination of ready-to-eat salads to the authorities after detecting the pathogen through its in-house routine quality control. Product and environmental samples collected during subsequent investigations yielded isolates, matching the outbreak strain, thus confirming that ready-to-eat salad from this company was most likely the outbreak source. The cause for the product contamination was related to a design-inherent hygienic problem of one specific product-feeding belt. Complementary patient interviews also identified ready-to-eat green salads bought at one retailer as the likely outbreak source.

Characterization of meticillin-resistant and meticillin-susceptible isolates of Staphylococcus pseudintermedius from cases of canine pyoderma in Australia

Meticillin-resistant Staphylococcus pseudintermedius (MRSP) has recently emerged as a worldwide cause of canine pyoderma. In this study, we characterized 22 S. pseudintermedius isolates cultured from 19 dogs with pyoderma that attended a veterinary dermatology referral clinic in Australia in 2011 and 2012. Twelve isolates were identified as MRSP by mecA real-time PCR and phenotypic resistance to oxacillin. In addition to β-lactam resistance, MRSP isolates were resistant to erythromycin (91.6 %), gentamicin (83.3 %), ciprofloxacin (83.3 %), chloramphenicol (75 %), clindamycin (66 %), oxytetracycline (66 %) and tetracycline (50 %), as shown by disc-diffusion susceptibility testing. Meticillin-susceptible S. pseudintermedius isolates only showed resistance to penicillin/ampicillin (90 %) and tetracycline (10 %). PFGE using the SmaI restriction enzyme was unable to type nine of the 12 MRSP isolates. However the nine isolates provided the same PFGE pulsotype using the Cfr91 restriction enzyme. Application of the mec-associated direct repeat unit (dru) typing method identified the nine SmaI PFGE-untypable isolates as dt11cb, a dru type that has only previously been associated with MRSP sequence type (ST)45 isolates that possess a unique SCCmec element. The dt11cb isolates shared a similar multidrug-resistant antibiogram phenotype profile, whereas the other MRSP isolates, dt11a, dt11af (dt11a-associated) and dt10h, were resistant to fewer antibiotic classes and had distinct PFGE profiles. This is the first report of MRSP causing pyoderma in dogs from Australia. The rapid intercontinental emergence and spread of multidrug-resistant MRSP strains confirms the urgent need for new treatment modalities for recurrent canine pyoderma in veterinary practice.

Identification of a marker for two lineages within the GC1 clone of Acinetobacter baumannii

Identification of a marker for two lineages within the GC1 clone of Acinetobacter baumannii

Empyema caused by MRSA ST398 with Atypical Resistance Profile, Spain

Empyema caused by MRSA ST398 with Atypical Resistance Profile, Spain

Prevalent genotypes of meticillin-resistant Staphylococcus aureus: report from Pakistan

Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and is emerging in the community. This is one of the first reports of the prevalent genotypes of MRSA in both hospital and community settings in Pakistan. Isolates collected in 2006-2007 were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). PFGE identified nine pulsotypes, the majority of isolates belonging to pulsotypes A (n=70) and B (n=38), which were predominant among hospital-onset MRSA (HO-MRSA) and community-onset MRSA (CO-MRSA) isolates, respectively. Among the HO-MRSA isolates, variants of SCCmec type III were prevalent, whilst SCCmec type IV or variants were predominant in the CO-MRSA isolates. MLST identified two principal sequence types, ST8 and ST239. An association was observed between ST8, PFGE pulsotype B and SCCmec type IV in the CO-MRSA (ST8-MRSA-IV). Similarly, ST239, PFGE pulsotype A and SCCmec type III were associated with HO-MRSA (ST239-MRSA-III). Therefore, the prevalent genotypes circulating in Pakistan at the time of study were ST8-MRSA-IV and ST239-MRSA-III in the community and hospital settings, respectively. A set of HO-MRSA isolates collected in 1997 were characterized by PFGE and SCCmec typing for comparison. The isolates belonged to two PFGE pulsotypes (A, n=28; B, n=11) and contained just two SCCmec types. These results suggest that an increase in genetic diversity occurred over the period 1997-2007 as a result of either microevolution or the importation of strains from surrounding areas.

Diversity of carbapenem resistance mechanisms in Acinetobacter baumannii from a Taiwan hospital: spread of plasmid-borne OXA-72 carbapenemase

We investigated the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii from a Taiwanese hospital and determined the mechanisms responsible for resistance. Ninety-two consecutive meropenem-resistant A. baumannii isolates collected between January 2005 and June 2007 were screened for genes encoding OXA carbapenemases, metallo-beta-lactamases and for the carO gene encoding an outer membrane protein. PFGE was used to define clonal relatedness. PCR mapping was used to examine the linkage of insertion sequences and bla(OXA) genes. Southern hybridization of plasmid extracts and I-CeuI-restricted chromosomal DNA was used to locate bla(OXA-24-like) genes. Sequences of selected bla(OXA-24-like) and carO genes were determined and loss of CarO expression was confirmed by SDS-PAGE. Most (70/92, 76%) isolates belonged to one of three PFGE pulsotypes, indicating clonal spread. Fifty-nine isolates, including the majority of those of pulsotypes I and III, produced OXA-72 carbapenemase. The bla(OXA-72) gene was located on a 54 kb plasmid in selected isolates. Thirty-three (36%) isolates, including all 16 of pulsotype II, had ISAba1 preceding the bla(OXA-51-like) gene, promoting its expression. In addition to OXA-72 carbapenemase, two pulsotype I and three pulsotype III isolates did not produce CarO protein as the carO gene was disrupted by insertion of an ISAba1 element. Two isolates of a minor pulsotype had a bla(OXA-58-like) gene and a single PFGE-unique isolate had a bla(OXA-23-like) gene. Although diverse mechanisms were identified, production of OXA-72 carbapenemase was the most common mechanism of carbapenem resistance in A. baumannii from this Taiwanese hospital. The plasmidic location of the gene had facilitated its spread to multiple strains.

Genotyping of Cronobacter (Enterobacter sakazakii) strains isolated from an infant formula processing plant.

Cronobacter spp. are occasional contaminants of infant food processing establishments and have been implicated in rare cases of neonatal infections. The control of these organisms during the processing of infant formula and identification of possible contaminating sources is of importance to manufacturers. In this study, infant formula processing sites were monitored for the presence of Cronobacter spp. (E. sakazakii) in 2007. Cronobacter were isolated using ISO/TS 22964 and simultaneously using a newly developed differential method, Cronobacter Screening Broth (CSB). Clonal types of Cronobacter strains in the environment, raw materials, and products were investigated using PFGE and RAPD in order to shed light on the possible dissemination routes within the facility. Over the course of the study, a total of 153 Cronobacter isolates were obtained from 46 end-products, 23 raw materials and 44 environmental samples. The 153 Cronobacter isolates represented 71 PFGE pulso-types, with 24 pulso-types containing multiple isolates. Clonal isolates present in products could be matched to indistinguishable pulso-types in raw materials and in the environment, however no strains were identified that were present in both the environment and raw materials. Although the majority of the isolates were obtained from products, the largest diversity of isolates was observed in raw materials. This may indicate raw materials as a significant source of Cronobacter entry into the production facility with subsequent selection for strains that are able to persist within the factory environment.