Comparison of quantitative PCR and MALDI-TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis.

AIMS: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS® ) after bacteriological growth. METHODS AND RESULTS: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2·105 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof® Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono- or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono- and bi-bacterial samples were not significantly different (McNemar test; P = 0·395) with a fair agreement (Cohen's kappa test; k = 0·375; P = 0·055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. CONCLUSIONS: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono- and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milk-associated bacterial species yet. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples.

Low prevalence of the 'gang of seven' and absence of the O80:H2 serotypes among Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) in intestinal contents of healthy cattle at two slaughterhouses in Belgium in 2014.

AIMS: The purpose of this survey was to estimate the respective prevalence of the 'gang of seven' and 'non-gang of seven' serotypes of Shigatoxigenic and enteropathogenic Escherichia coli and to identify the O80:H2 serotype in 245 intestinal contents collected at two slaughterhouses in Belgium in 2014. METHODS AND RESULTS: After overnight enrichment growth, the 69 intestinal contents testing positive with PCR targeting the eae, stx1 and stx2 genes were inoculated onto four agar media. Of the 2542 colonies picked up, 677 from 59 samples were PCR confirmed. The most frequent virulotypes were eae+ in 47 (80%) samples, stx2+ in 20 (34%) samples and eae+ stx1+ in 16 (27%) samples. PCR-positive colonies belonged to different virulotypes in 36 samples. No colony was O80-positive, whereas two eae+ colonies from two samples were O26:H11, 50 eae+ stx1+ and eae+ from eight samples were O103:H2 and two eae+ stx1+ stx2+ colonies from one sample were O157:H7. CONCLUSIONS: The 'non-gang of seven' serotypes are more frequent than the 'gang of seven' serotypes and the O80:H2 serotype was not detected among Shigatoxigenic and enteropathogenic Escherichia coli in the intestines of cattle at these two slaughterhouses. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the identification protocols of Shigatoxigenic Escherichia coli focus on the 'gang of seven' serotypes, several other serotypes can be present with possible importance in public health. Innovative selective identification procedures should be designed.

Comparison of culture-dependent and -independent bacterial detection results on nasal swabs in dogs with nasal discharge.

OBJECTIVES: The role of bacterial communities in the pathophysiology of canine nasal disease is still unclear. How and when to treat dogs with suspected secondary bacterial rhinitis and on which test to rely before making a decision to treat with antimicrobials has not been established. The objective is to compare the results of bacterial identification using agar-plate cultures and 16S rRNA gene amplicon sequencing in dogs with nasal discharge suspected to be of bacterial origin. MATERIALS AND METHODS: Twenty-nine client-owned dogs presented for investigation of nasal disease were included in the study. Paired swabs were collected from the same affected nasal cavity. One swab was streaked on 4 agar media (Columbia Blood Agar, MacConkey, Chapman and Edward's). The other swab was stored in a sterile cryotube at -80°. Extracted DNA underwent a polymerase chain reaction targeting the V1-V3 region of the 16S rRNA gene. RESULTS: At least one of the species detected by amplicon sequencing with a relative abundance of >10% was also identified by culture in 14 cases (48.3%), in association with marked predominance of one taxon (>80% relative abundance) in six of 14 cases. In 12 dogs (41.4%), the cultured isolates were rare or undetected components of the corresponding sequence libraries. A negative culture in the face of bacterial predominance (>50% relative abundance) of a potentially pathogenic bacteria detected by sequencing occurred in 17% (n=5) of cases; however, the use of other agar media may have decreased this percentage. CLINICAL SIGNIFICANCE: Standard culture does not reliably predict the bacterial profile detected by 16S rRNA gene amplicon sequencing.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk of bovine mastitis.

The aim of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus (MRSA) among a (S. aureus) collection (n = 430) isolated from milk of cows suffering from mastitis in Belgium and to compare their genotypic as well as phenotypic characteristics. Pulsed field gel electrophoresis (PFGE) and PCR-based typing techniques (MLST, spa, SCCmec, and agr typing) have been applied and supplemented by capsule serotyping, biofilm production quantification and antimicrobial susceptibility testing. Nineteen MRSA were isolated. Seven distinct ApaI PFGE patterns were observed. All isolates, except one, were identified as ST398 strains. Three spa types (t011, t567 and t108) and two SCCmec types (IV and V) were identified. All isolates belonged to agr type I and capsule type 5 and were Panton-Valentine leukocidin (PVL) negative. All isolates produced biofilm in TSBglc , whereas the majority did not in milk serum. Twelve resistance patterns were observed, with almost two-thirds of the isolates being resistant to at least six antibiotics, including penicillin and tetracycline. Our study confirms that the emerging ST398 LA-MRSA clone has attained Belgian cattle. With regard to genotypic and phenotypic typing, the 19 MRSA isolated in this study form a homogenous group and do not differ much from one another, neither from what has been previously described.

Enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli in wild cervids.

AIMS: The aim of this study was to investigate the presence of enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli strains in free-ranging wild ruminants in Belgium and to characterize the positive isolates (serogroups and virulence-associated factor-encoding genes). METHODS AND RESULTS: Escherichia coli strains isolated from faeces of wild cervids were characterized by PCR targeting genes coding for the main virulence properties of EPEC, EHEC and VTEC strains. The prevalence rate of these pathogenic strains in faecal samples obtained from the wild ruminants was found to be 15%. No pathogenic isolate was found to belong to the O157, O26, O111, O103 or O145 serogroups. Moreover, a new gene, eibH, showing 88% identity with eibG was detected in VTEC strains. CONCLUSIONS: The results reveal that wild ruminants could be considered as a potential source of VTEC and EPEC infection for humans and possibly also for domestic ruminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests the potential risk of transmission of VTEC, EHEC and EPEC strains from wild ruminants to humans via the consumption of venison and to domestic ruminants because of sharing of the same pasture. Indeed, many serogroups other than O157 EHEC have also been shown to be responsible for outbreaks in humans in several countries, and studies focusing solely on O157:H7 EHEC tend to underestimate this risk of transmission.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC.

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar.

Mediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections.

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log2-transformed CFU that elicited one unit increase in log2-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log2-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components.