Antimicrobial susceptibility profile of Klebsiella pneumoniae isolated from some dairy products in Libya as a foodborne pathogen.

Background and Aim: Klebsiella pneumoniae is one of the most common causes of clinical and asymptomatic mastitis in dairy cattle, as well as in milk and dairy products that affect milk quality. Mastitis caused by K. pneumoniae is even more serious due to its poor response to antibiotic therapy. The aim of this study was to detect and identify the presence of K. pneumoniae in milk and dairy products produced in Libya. Materials and Methods: A total of 234 samples were randomly collected from various locations in Libya. Samples were examined for the presence of K. pneumoniae using conventional cultural techniques, including cultivation in violet red bile agar plus 4-methylumbelliferyl-ß-D-glucuronide broth and CHROM agar, followed by polymerase chain reaction identification and partial sequencing of 16S rRNA. Results: Of the 234 samples of milk and dairy products collected, 16 (6.8%) isolates revealed mucoid colonies on agar media that were phenotypically suggested to be K. pneumoniae. Identification of isolates was confirmed using molecular techniques (16S rRNA). Among the examined samples, K. pneumoniae was recovered from camel's milk, raw cow's milk, raw fermented milk, Maasora cheese, Ricotta cheese, soft cheese, full cream milk powder, milk powder infant formula, cereal baby food, and growing-up formula. Antibiotic susceptibility testing was performed on 12 of the 16 K. pneumoniae isolates, and the results showed that K. pneumoniae isolates were resistant to more than eight antibiotics; interestingly, two isolates showed metallo-beta-lactamase (MBL) production. Conclusion: K. pneumoniae is considered a risk to human health because many of these products do not comply with the microbiological criteria of international and/or Libyan standards. This study emphasized the relationship between K. pneumoniae and raw milk, cheese, milk powder, and infant milk retailed in Libya. There is a need to take the necessary measures to ensure effective hygiene practices during production in dairy factories, handling, and distribution on the market, in particular at a small local production scale.

Antibacterial activity of flavonoid extracts from Enteromorpha intestinalis and Caulerpa prolifera against multidrug-resistant foodborne bacterial isolates.

Background: Food poisoning caused by bacterial agents is a worldwide problem, usually accompanied by unpleasant symptoms and may be severe leading to death. Natural compounds from marine algae namely flavonoids may play a role in the remedy of this condition. Aim: This research aims to assess the potency of flavonoids extracted from Enteromorpha intestinalis and Caulerpa prolifera as antibacterial agents. Methods: Enteromorpha intestinalis was collected from Western Libyan Coast and C. prolifera was collected from Farwa Island. The antimicrobial activity and determination of minimum inhibitory concentration of algal flavonoid-containing extracts was performed in vitro against some positive and negative Gram bacteria. Results: Crude extract containing flavonoids from E. intestinalis was more effective than C. prolifera extract against Staphylococcus aureus with antimicrobial essay (25-28 + 1 and 14.5-37.5 + 0.5-1.5), MIC (50 and 50-250 µg/ml), MBC (75 and 75-250 µg/ml). In Bacillus cereus, the antimicrobial assay (19-24.5 + 0.5-1.5: 24 + 1), MIC (50-250 + 100 µg/ml), and MBC (250 and 125 µg/ml). On the other hand, flavonoids containing extract from C. prolifera were more effective than E. intestinalis against Enterohemorrhagic Escherichia coli O157 EHEC O157 (25-28 + 1: 14-18.5 + 0.5-1.5), MIC (100-250:100-500 µg/ml), and MBC (150-250 and 250-500 µg/ml). Salmonella enterica qualitatively combat by flavonoid from E. intestinalis (13.5-14 + 0.5-1: 10.5-13.5 + 0.5-1.5), MIC (100-250: 250 µg/ml), and MBC (100-250: 250 µg/ml). Flavonoids from C. prolifera (4 strains: 2 strains) were effective against S. enterica. Crude flavonoids from both algae were not effective against Bacillus pumilus. Conclusion: Data from this study could conclude that flavonoid extracts from E. intestinalis and C. prolifera could be used against foodborne bacterial agents.

Thermal tolerance of Cronobacter sakazakii and Cronobacter pulveris in reconstituted infant milk formula.

Background: Cronobacter sspecies are the most significant foodborne pathogen in infant milk formula (IMF). These pathogens have been incriminated in severe forms of neonatal meningitis, sepsis, and necrotizing enterocolitis with a high mortality rate. Aim: This study was performed to elucidate the effect of heat stress on Cronobacter spp. (C. sakazakii and C. pulveris) in reconstituted IMF (RIMF). Methods: The reconstituted formula was inoculated with five C. sakazakii isolates and four C. pulveris isolates separately. The nine isolates of Cronobacter spp. were heated in RIMF at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C. The D- and z-values were determined by using linear regression analysis. Results: The D-values of all isolates of C. sakazakii (CS1, CS3, CS4, CS5, and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C were in the ranges 7.29-23.47, 2.77-15.50, 0.62-1.04, 0.62-1.02, 0.62-1.00, 0.62-1.00 minutes, respectively; while, the z-values extended from 2.50°C to 4.28°C. The D- values of C. pulveris isolates (CP1, CP2, CP3, CP4) were in the ranges 7.60-22.32, 1.42-8.45, 0.62-1.08, 0.62-0.78, 0.62-0.78, 0.62-0.79 minutes at 48°C, 52°C, 56°C, 60°C, 64°C, 66°C, respectively and the calculated z-values ranged from 3.33°C to 4.89°C. Conclusion: This study may contribute to improving the understanding of the behavior of C. sakazakii and C. pulveris isolates in RIMF at various heat stress temperatures and may participate in the effective control of these pathogens in infant food production.

Occurrence and antibiogram of multidrug-resistant Salmonella enterica isolated from dairy products in Libya.

Background and Aim: Foodborne illnesses are a serious challenge to human health and the economic sector. For example, salmonellosis remains a burden in developed and developing nations. Rapid and reliable molecular methods to identify Salmonella strains are essential for minimizing human infection. This study aimed to identify Salmonella spp. in raw milk and dairy products using conventional and molecular techniques and to test the antibiotic susceptibility of the isolated strains. Materials and Methods: One hundred and thirty-one milk and dairy product samples were randomly collected from different localities in Libya. Samples were examined for the presence of Salmonella by conventional culture techniques, including cultivation in Rappaport-Vassiliadis broth and streaking on xylose lysine deoxycholate agar. Identification also used polymerase chain reaction and partial sequencing of 16S rDNA. Twenty-four antibiotics were used for the examination of antimicrobial resistance of Salmonella spp. isolates with the agar disk diffusion method (Kirby-Bauer technique). Multi-antibiotic resistance index and antibiotic resistance index (ARI)for Salmonella enterica isolates were calculated. Results: Twenty-one of 131 samples (16%) were positive for Salmonella spp. recovered from 9 (16%), 2 (11%), 4 (22.2%), and 6 (46%) samples of raw cow milk, fermented raw milk, and fresh locally made soft cheeses, Maasora and Ricotta), respectively. Samples of ice cream, milk powder, and infant formula showed no Salmonella spp. contamination. Only 9 of 21 (42.8%) isolates were confirmed as S. enterica by partial sequence 16S rDNA analysis. All isolates were resistant to amoxycillin, bacitracin, penicillin G, lincomycin, vancomycin, clindamycin, and cloxacillin with an ARI of 0.042. In contrast, all tested strains were sensitive to levofloxacin, doxycycline, and ciprofloxacin. In addition, all of the tested isolates (100%) were resistant to more than one antibiotic. Conclusion: This study demonstrated the applicability of molecular techniques, compared with conventional methods, as preferable for the identification of Salmonella in milk and dairy products and thus reduction of milk-borne transmission to the consumers. From the view of public health, isolation and identification of Salmonella multidrug-resistant strains from raw cow`s milk and locally prepared dairy products sold in the Libyan markets indicate the need to improve the handling and processing of milk and dairy products to minimize the prevalence of Salmonella, one of the most important foodborne microorganisms that cause food poisoning.

Extent of pathogenic and spoilage microorganisms in whole muscle meat, meat products and seafood sold in Libyan market.

Background: Whole muscle meat, meat products, and seafood contain different nutrients in adequate quantity providing a better environment for presence and replication of different microorganisms. There are underreported and inaccurate estimations of foodborne diseases due to the lack of effective surveillance systems in Libya. Aim: To determine the extent of microbiological contamination of whole muscle meat, meat products, and seafood. Methods: A total number of 731 samples of retail meat were collected from different stores in four cities in Libya. Samples were analyzed for aerobic plate count and subjected to microbiological enumeration and isolation techniques, followed by molecular identification by PCR and partial sequencing of 16S rDNA. Results: The results showed contamination of samples with enteric and spoilage bacteria. Fifteen genera of spoilage bacteria yielded 149 isolates which were detected and identified by PCR and partial sequencing of 16S rDNA as: Proteus spp., Provedencia spp., Raouttella ornithinolytical, Citrobacter spp., Enterobacter spp., Morganella morgi, Shewanella algea, Rhodobacter capsulatus, Listonella pelagia, Kluyvera spp., Pectobacterium spp., Brenneria spp., Klebsiella spp., Acintobacter radioresistens, and Pantoea spp. While for pathogenic bacteria, 143 isolates distributed among nine genera were identified by PCR and partial sequencing of 16S rDNA as: Bacillus spp., Escherichia spp., Shigella spp., Enterococci spp., Cronobacter spp., Staphylococci spp., Salmonella spp., Aeromonas spp., and Vibrio spp.. Many isolated bacteria are zoonotic bacteria with high importance for public health. Conclusion: Excessive handling and processing of meat and meat products seems to be one of the poorest microbiological qualities. These findings ought to be helpful in risk assessments and quality assurance of meat in order to improve food safety.

Occurrence, characterization, and antibiogram of Staphylococcus aureus in meat, meat products, and some seafood from Libyan retail markets.

AIM: The aim of the current investigation was to screen the presence of Staphylococci spp., especially S. aureus in meat, meat products of different animal species, and some seafood sold in some retail markets in Libya using cultural and molecular techniques, and to study their antibiotics resistance profiles. MATERIALS AND METHODS: A total of 139 samples from red meat, meat products, and seafood were collected from many areas in Libya. Enumeration and isolation of Staphylococci spp. and S. aureus by normal cultural methods followed by molecular identification using molecular techniques by bacterial DNA extraction and partial sequencing of 16S rDNA. RESULTS: Out of 139 samples, 112 (80.6%) were contaminated with different species of Staphylococci based on cultural characteristics of Staphylococci on Baird-Parker medium, for which S. aureus was detected in only 32 samples (23%). However, only six out of 18 (33.3%) isolates sent for sequencing were confirmed to be S. aureus using the molecular technique. The six identified isolates of S. aureus were tested for antimicrobial resistance against 24 most commonly used antibiotics. All isolates were resistant to only two antibiotics (cefotaxime and clindamycin). Among these six isolates, only one confirmed to be Methicillin-resistant Staphylococcus aureus. CONCLUSION: Results of this study suggest that food of animal origin could be a source of S. aureus with antimicrobial resistance characteristics that can be spread through the food chain, and raise the importance of these results for public health.

Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA.

AIM: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. MATERIALS AND METHODS: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow's milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. RESULTS: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow's milk (11%), 3 isolates from she-camel's milk (11%), two isolates from goat's milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. CONCLUSION: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.